U.S. patent application number 16/715692 was filed with the patent office on 2020-06-18 for compositions and methods for cancer immunotherapy.
The applicant listed for this patent is The UAB Research Foundation Incysus Therapeutics, Inc.. Invention is credited to William Ho, JR., Lawrence S. Lamb, JR..
Application Number | 20200188436 16/715692 |
Document ID | / |
Family ID | 61197093 |
Filed Date | 2020-06-18 |
United States Patent
Application |
20200188436 |
Kind Code |
A1 |
Ho, JR.; William ; et
al. |
June 18, 2020 |
Compositions and Methods for Cancer Immunotherapy
Abstract
The present invention provides compositions and methods for
combination therapy comprising administering to a patient in need
thereof, drug-resistant immunotherapy, immune checkpoint
inhibitors, and chemotherapy for the treatment of cancer.
Inventors: |
Ho, JR.; William; (New York,
NY) ; Lamb, JR.; Lawrence S.; (Birmingham,
AL) |
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Applicant: |
Name |
City |
State |
Country |
Type |
The UAB Research Foundation
Incysus Therapeutics, Inc. |
Birmingham
New York |
AL
NY |
US
US |
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|
Family ID: |
61197093 |
Appl. No.: |
16/715692 |
Filed: |
December 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16278336 |
Feb 18, 2019 |
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16715692 |
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PCT/US2017/047515 |
Aug 18, 2017 |
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16278336 |
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62376680 |
Aug 18, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/0011 20130101;
C12N 2506/45 20130101; A61K 35/17 20130101; A61K 45/06 20130101;
A61P 37/02 20180101; C12Y 201/01063 20130101; C12N 15/52 20130101;
A61K 2039/505 20130101; C12N 5/0696 20130101; C12Y 105/01003
20130101; C07K 14/705 20130101; C07K 16/2827 20130101; C07K 16/2818
20130101; A61P 35/00 20180101; C12N 2510/00 20130101; A61K 38/44
20130101; A61P 43/00 20180101; A61K 38/45 20130101; C12Y 206/01044
20130101; C12N 5/0638 20130101; A61K 39/3955 20130101; A61K 31/495
20130101; C12Y 201/01045 20130101; A61K 38/177 20130101; A61K
31/495 20130101; A61K 2300/00 20130101; A61K 35/17 20130101; A61K
2300/00 20130101; A61K 39/3955 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C12N 15/52 20060101 C12N015/52; C12N 5/074 20060101
C12N005/074; C12N 5/0783 20060101 C12N005/0783; C07K 14/705
20060101 C07K014/705; A61K 39/00 20060101 A61K039/00 |
Claims
1. A method for the treatment of cancer in a patient in need
thereof comprising the steps of: i. obtaining a population of
isolated cytotoxic immune cells comprising .gamma..delta. T-cells,
natural killer (NK) cells, or any combination thereof, wherein the
population of cytotoxic immune cells have been genetically modified
to be resistant to one or more therapeutic agents; ii.
administering to the patient an effective amount of one or more of
the therapeutic agents to which the genetically modified cytotoxic
immune cells of step (i) are resistant; iii. administering to the
patient the population of genetically modified cytotoxic immune
cells of step (i); and iv. administering to the patient an
effective amount of at least one immune checkpoint inhibitor.
2. The method of claim 1, wherein the cancer is selected from
glioma, glioblastoma, lymphoma, melanoma, neuroblastoma, non-small
cell lung cancer, renal cell carcinoma, and small cell lung
cancer.
3. The method of claim 2, wherein the cancer is glioma,
glioblastoma, or neuroblastoma.
4. The method of claim 1, wherein the isolated cytotoxic immune
cells comprise .gamma..delta. T-cells, NK cells and further
optionally comprise other immunocompetent cells.
5. The method of claim 4, wherein the isolated cytotoxic immune
cells have been genetically modified to encode alkyl guanine
transferase (AGT), P140KMGMT, O.sup.6 methylguanine DNA
methyltransferase (MGMT), L22Y-DHFR, thymidylate synthase,
dihydrofolate reductase, or multiple drug resistance-1 protein
(MDR1).
6. The method of claim 5, wherein the isolated cytotoxic immune
cells have been genetically modified to encode P140KMGMT.
7. The method of claim 1, wherein the immune checkpoint inhibitor
targets CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3,
GAL9, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55),
CGEN-15049, CHK 1 kinase, CHK26 kinase, A2aR, OX40, or a B-7 family
ligand.
8. The method of claim 7, wherein the checkpoint inhibitor targets
PD-1, PDL1, PDL2 or CTLA-4.
9. The method of claim 1, wherein the immune checkpoint inhibitor
is selected from Tremelimumab, anti-OX40, PD-L1 monoclonal Antibody
(Anti-B7-H1; MEDI4736), MK-3475, OPDIVO.RTM./Nivolumab, CT-011,
BY55 monoclonal antibody, AMP224, BMS-936559, MPLDL3280A,
MSB0010718C, YERVOY.RTM./ipilimumab, and pembrolizumab
(KEYTRUDA.RTM.).
10. The method of claim 1, wherein the therapeutic agent is
selected from an alkylating agent; a metabolic antagonist; a DNA
demethylating agent; a substituted nucleotide; a substituted
nucleoside; an antitumor antibiotic; a plant-derived antitumor
agent; cisplatin; carboplatin; etoposide; methotrexate (MTX);
trimethotrexate (TMTX); temozolomide; raltitrexed;
S-(4-Nitrobenzyl)-6-thioinosine (NBMPR); 6-benzyguanidine (6-BG); a
nitrosourea; cytarabine; camptothecin; and a therapeutic derivative
of any thereof.
11. The method of claim 1, wherein the isolated cytotoxic immune
cells have been genetically modified to be resistant to two
therapeutic agents selected from an alkylating agent; a metabolic
antagonist; a DNA demethylating agent; a substituted nucleotide; a
substituted nucleoside; an antitumor antibiotic; a plant-derived
antitumor agent; cisplatin; carboplatin; etoposide; methotrexate
(MTX); trimethotrexate (TMTX); temozolomide; raltitrexed;
S-(4-Nitrobenzyl)-6-thioinosine (NBMPR); 6-benzyguanidine (6-BG); a
nitrosourea; cytarabine; camptothecin; and a therapeutic derivative
of any thereof.
12. The method of claim 11, wherein the two therapeutic agents are
temozolomide and methotrexate.
13. The method of claim 11, wherein the isolated cytotoxic immune
cells have been genetically modified with the drug resistant genes
alkyl guanine transferase (AGT) and dihydrofolate reductase.
14. The method of claim 1, wherein administration of the
genetically modified immune cells of step (iii) and the
administration of the checkpoint inhibitors of step (iv) occurs
substantially simultaneously or sequentially.
15. A composition comprising at least one checkpoint inhibitor, and
an isolated population of cytotoxic immune cells comprising
.gamma..delta. T-cells, NK cells, or any combination thereof,
wherein greater than about 50% of the population of cytotoxic
immune cells express a polypeptide that confers resistance to a
chemotherapy agent.
16. The composition of claim 15, wherein the isolated population of
cytotoxic immune cells comprises about 50% to about 95%
.gamma..delta. T-cells and comprises about 5% to about 25% NK
cells.
17. The method of claim 15, wherein the checkpoint inhibitor
targets PD-1, PDL1, PDL2 or CTLA-4.
18. The method of claim 15, wherein the cytotoxic immune cells have
been genetically modified to encode alkyl guanine transferase
(AGT), P140KMGMT, O.sup.6 methylguanine DNA methyltransferase
(MGMT), L22Y-DHFR, thymidylate synthase, dihydrofolate reductase,
or multiple drug resistance-1 protein (MDR1).
19. The method of claim 1, wherein the isolated cytotoxic immune
cells are derived from human induced pluripotent stem cells
(hiPSCs).
20. The method of claim 19, wherein the isolated cytotoxic immune
cells comprise .gamma..delta. T cells.
21. The method of claim 20, wherein the isolated cytotoxic immune
cells further comprise NK cells.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/278,336, filed Feb. 18, 2019, which is a continuation of
PCT/US2017/047515, filed on Aug. 18, 2017, which designated the
United States, published in English, which claims the benefit of
U.S. Provisional Application No. 62/376,680, filed on Aug. 18,
2016. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Although outstanding progress has been made in the fields of
cancer detection and therapy, the treatment of late-stage and
metastatic cancer remains a major challenge. Cytotoxic chemotherapy
agents remain among the most used and successfully employed
anti-cancer treatments. However, they are not uniformly effective,
and the introduction of these agents with novel therapies, such as
immunotherapies, is problematic. For example, chemotherapy agents
can be detrimental to the establishment of robust anti-tumor
immunocompetent cells due to the agents' non-specific toxicity
profiles. Small molecule-based therapies targeting cell
proliferation pathways may also hamper the establishment of
anti-tumor immunity. Further complications found in
immunotherapeutic approaches is the ability of tumor cells to
outsmart the body's immune response through the downregulation of
MHC-class I antigen expression, presentation of immune checkpoint
molecules on the tumor or effector cell surfaces or the shedding of
soluble tumor ligands into the plasma leading to effector cell
receptor downregulation. Such processes lead to a tumor that
appears normal resulting in the evasion of an immune system attack.
However, if chemotherapy regimens that are transiently effective
can be combined with both novel immunocompetent cell therapies and
immune checkpoint inhibitors, then significant improvement in
anti-neoplastic therapy might be achieved.
[0003] Several drug resistant genes have been identified that can
potentially be used to confer drug resistance to targeted immune
cells, and advances in gene therapy techniques have made it
possible to test the feasibility of using these genes in drug
resistance gene therapy studies. For example, an shRNA strategy was
used to decrease the levels of hypoxanthine-guanine
phosphoribosyltransferase (HPRT), which conferred resistance to
6-thioquanine. Also, the drug resistant gene MGMT encoding human
alkyl guanine transferase (hAGT) is a DNA repair protein that
confers resistance to the cytotoxic effects of alkylating agents,
such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG)
is an inhibitor of AGT that potentiates nitrosourea toxicity and is
co-administered with TMZ to potentiate the cytotoxic effects of
this agent. Several mutant forms of MGMT that encode variants of
AGT are highly resistant to inactivation by 6-BG, but retain their
ability to repair DNA. P140KMGMT-based drug resistant gene therapy
has been shown to confer chemoprotection to mouse, canine, rhesus
macaques, and human cells, specifically hematopoietic cells.
[0004] Tumor cells often express cell-surface molecules that reveal
their cancerous nature. However, these same cells may also present
immune checkpoint molecules on their surfaces that mimic those of
normal cells, thereby avoiding an immune attack. Immune checkpoints
are often regulated by interactions between specific
receptor/ligand pairs including CTLA-4 and PD-1. CTLA-4, PD-1 and
its ligands are members of the CD28-B7 family of co-signaling
molecules that play important roles throughout all stages of T-cell
function and other cell functions. The PD-1 receptor is expressed
on the surface of activated T cells (and B cells) and, under normal
circumstances, binds to its ligands (PD-L1 and PD-L2) that are
expressed on the surface of antigen-presenting cells, such as
dendritic cells or macrophages. This interaction sends a signal
into the T cell and essentially switches it off or inhibits it.
Cancer cells take advantage of this system by driving high levels
of expression of PD-L1 on their surface. This allows them to gain
control of the PD-1 pathway and switch off T cells expressing PD-1
that may enter the tumor microenvironment, thus suppressing the
anticancer immune response.
[0005] Checkpoint inhibitors provide a means to unmask tumor cells
by blocking receptor/ligand pairs such as CTLA-4 or PD-1, allowing
the T cells to mount an immune response. Six immune checkpoint
inhibitors that have received accelerated approval from the U.S.
Food and Drug Administration for cancer are ipilimumab
(YERVOY.RTM.), pembrolizumab (KEYTRUDA.RTM.), atezolizumab
(TECENTRIQ.RTM.), durvalumab (IMFINZITM), avelumab (BAVENCIO), and
nivolumab (OPDIVO.RTM.). YERVOY.RTM. is a monoclonal antibody that
targets CTLA-4 on the surface of T cells and is approved for the
treatment of melanoma. KEYTRUDA.RTM. targets PD-L1 and is used to
treat melanoma and non-small cell lung cancer. OPDIVO.RTM. also
targets PD-1 and is approved for treatment of melanoma, renal cell
carcinoma, and non-small cell lung cancer. Additional checkpoint
targets that may prove to be effective are TIM-3, LAG-3, various
B-7 ligands, CHK 1 and CHK2 kinases, BTLA, A2aR, and others.
[0006] Typically, activity and high objective response rates (ORR)
in connection with checkpoint inhibitor treatment is associated
with tumors with high mutational loads. Mutation frequency is
generally correlated with high neoantigen signaling resulting in
increased tumor immunogenicity.
[0007] In May 2017, The U.S. Food and Drug Administration (FDA)
granted accelerated approval to pembrolizumab, for adult and
pediatric patients with unresectable or metastatic, microsatellite
instability-high (MSI-H) or mismatch repair deficient (dMMR) solid
tumors that have progressed following prior treatment and who have
no satisfactory alternative treatment options or with MSI-H or dMMR
colorectal cancer that has progressed following treatment with a
fluoropyrimidine, oxaliplatin, and irinotecan. This is the FDA's
first approval for a tissue/site agnostic indication. Patients with
dMMR have a defect that confers an inability to correct genetic
mutations that leads to a hypermutated state.
[0008] Clinical data has demonstrated that in cancers such as
colorectal cancer where the ORR is typically 0% with checkpoint
inhibitor treatment, ORR's are increased to 62% in those with dMMR
due to their high mutational burden (Le DT, et al. ASCO 2015.
Abstract LBA100). Similarly, an analysis by Hodges et al.
(Neuro-Oncology, 19(8), 1047-1057, 2017) demonstrates that
glioblastomas (GBMs) typically have a low mutation burden with only
3.5% of those GBMs analyzed demonstrating a high tumor mutational
load. In those GBM patients with dMMR, however, they have been
found to have the highest mutation load over other high-grade
tumors by Boufett et al. (JCO, 2016 34:19, 2206-2211). Boufett et
al. describe the remarkable and durable responses of two pediatric
patients with recurrent multifocal GBM who were refractory to
current standard therapies when treated with single-agent
nivolumab. A dMMR mutation drove these two patients to responses
with checkpoint inhibition, but their failed responses to standard
therapies may have also been due to dMMR. This supports data
suggesting that generally GBM's are not significantly immunogenic
and are not likely to demonstrate significant ORR's when treated
with checkpoint inhibitors.
[0009] Since 2005, standard-of-care treatment for front-line GBM
has been the Stupp Protocol, involving treatment with the
chemotherapy temozolomide (TMZ) (Stupp et al., N Engl J Med 2005;
352:987-996 Mar. 10, 2005). TMZ is an alkylating agent that causes
double stranded DNA breaks. TMZ functions by creating
O.sup.6-methylguanine (O.sup.6MeG) adducts in DNA which will cause
a mispairing and formation of a GC to AT point mutation (Roos et
al., Cancer Letters 332 (2013) 237). The resistance mechanism to
effective treatment with TMZ is
O.sup.6-methylguanine-methyltransferase (MGMT), which removes the
methyl adduct from the O.sup.6 position. In cells that have a
functioning MMR system, the GC to AT point mutation will cause a
double stranded break after two cycles of DNA replication. As Roos
notes, "O.sup.6MeG does not trigger apoptosis directly; it requires
MMR and DNA replication." Thus, those newly diagnosed GBM patients
who are most likely to respond to immunotherapy and/or checkpoint
inhibition due to high mutational load and neoantigen burden, are
also those least likely to respond to conventional alkylating
chemotherapies. This realization would explain the observation that
MMR and MLH1 mutations are actually a negative prognosticator for
survival in GBM patients (Draaisma et al., Acta Neuropathol Commun.
2015; 3: 88). These patients don't respond to conventional therapy
with TMZ.
[0010] The present invention provides a solution to this challenge.
While hypermutations due to dMMR can lead to stronger immunogeneic
signaling, they also likely result in low responses to standard
chemotherapeutic agents and low survival. By causing double
stranded-DNA breaks, TMZ has demonstrated the ability to drive
hypermutations in tumors and stronger immunogeneic signaling. By
artificially creating hypermutations with chemotherapy, one can
maintain tumor sensitivity to standard treatment while increasing
tumor immunogenicity and responsiveness to checkpoint inhibition if
one can also keep the lymphocytes alive during chemotherapy
treatment.
[0011] Unfortunately, over time TMZ driven hypermutations are
likely to cause mutations in the MMR system, leading to decreased
sensitivity to TMZ and other alkylating agents. Without being bound
by theory, the hypermutations caused by TMZ or other alkylating
agents are most likely to be subclonal signature 11 mutations that
may not elicit long term immunogenicity (Alexandrov et al., Nature,
Vol 500, 22 Aug. 2013). Shorter term however, our data demonstrates
that the initiation of the double stranded break can trigger a DNA
damage response that results in the transient upregulation of the
NKG2D receptor ligands on the tumor cell surface and trigger
activation of an innate immune response and tumor killing by drug
resistant .gamma..delta. T-cells (Lamb et al., PLOS ONE, Vol 8,
Issue 1, January 2013).
[0012] The present invention thus targets both mechanisms together
early on in the progression of cancer. Without being bound by
theory, the DNA damage caused by TMZ results in the upregulation of
ataxia telangiectasia mutated (ATM) and ataxia telangiectasia Rad-3
related (ATR) protein kinases associated to the DNA damage
response. The DNA damage response and upregulation of ATM and ATR
results in the upregulation of NKG2D ligands such as MICA/B and the
ULBP16 on the tumor cell surface and the initiation of an
anti-tumor immune response. By killing most of the tumor that is
sensitive to chemotherapy while upregulating the immune response
through DNA damage and taking the brakes off of the immune cells
with checkpoint inhibition, we seek to create stronger, more
durable responses for cancer patients.
[0013] Accordingly, there remains an urgent need for combination
therapies that enhance, replace or supplement current methods of
treating cancers, and in particular those cancers that exhibit
transient responses to chemotherapy. Combination therapies
comprising drug-resistant immunocompetent cells, immune checkpoint
inhibitors, and chemotherapy offers such a supplemental
approach.
SUMMARY OF THE INVENTION
[0014] The present invention provides combination therapies for
treating cancer comprising compositions and methods for enhancing
the immune response of immunocompetent cells against cancer,
including protection from drug-induced toxicities during
chemotherapy, thereby allowing for the combined administration of
immuno- and chemotherapy, an anticancer treatment termed "drug
resistant immunotherapy" in combination with one or more cycles
and/or doses of an immune checkpoint inhibitor. The combination of
checkpoint inhibitors and drug resistant immunotherapy may be
administered sequentially in any order, or substantially
simultaneously. Drug resistant-immunotherapy comprises genetic
modification of isolated cytotoxic immune cells. Preferably, the
isolated cytotoxic immune cells are genetically modified using any
method known in the art. Preferably the method of genetic
modification includes, but is not limited to, an HIV-based
lentiviral system or a gene editing system. Preferably the gene
editing system comprises the use of directed endonucleases,
including, but not limited to, Zinc Finger Nucleases (ZFNs),
Transcription Activator Like Effector Nucleases (TALENs), or
proteins, like Cas9, associated with Clustered Regularly
Interspaced Palindromic Repeats (CRISPR) to deliver the drug
resistance-conferring genetic element into immunocompetent cell
lines. Genetically engineered immunocompetent cells develop
significant resistance to a specific chemotherapeutic cytotoxic
agent compared to non-modified cells; however, the drug resistance
does not affect the genetically engineered cell's ability to kill
target cancer cells in the presence or absence of a chemotherapy
agent. Such drug resistant immunotherapy is described, for example,
by Spencer H. T. et al. in US 2015/0017137.
[0015] The present invention provides methods for treating cancer
in a patient, comprising the steps of: obtaining an optionally
enriched and/or optionally expanded population of cytotoxic immune
cells, wherein the cytotoxic immune cells comprise cells that have
been genetically modified to be resistant to a therapeutic agent;
administering to a patient in need thereof, an effective amount of
the therapeutic agent to which the genetically engineered cells are
resistant in combination with the population of genetically
modified cytotoxic immune cells, and further administering to the
patient an effective amount of at least one immune checkpoint
inhibitor, thereby treating cancer in the patient.
[0016] Preferably, the population of cytotoxic immune cells used in
the compositions and methods of the invention comprises
.gamma..delta. T-cells. Preferably the population of cytotoxic
immune cells comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
75%, 80%, 85%, 90%, 95% or 100% .gamma..delta. T-cells. Preferably
the population of cytotoxic immune cells comprises about 50% to
about 95% .gamma..delta. T-cells. Preferably the population of
cytotoxic immune cells used in the compositions and methods of the
invention comprises .gamma..delta. T-cells and natural killer (NK)
cells. Preferably the population of cytotoxic immune cells
comprises about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, or more of NK cells. Preferably the population of cytotoxic
immune cells comprises about 5% to about 25% of NK cells.
Preferably the population of cytotoxic immune cells used in the
compositions and methods of the invention comprise .gamma..delta.
T-cells, NK cells and other immunocompetent cells including, but
not limited to: monocytes, dendrites and macrophages. Preferably,
the population of cytotoxic immune cells used in the compositions
and methods of the invention comprise .gamma..delta. T-cells
derived from human induced pluripotent stem cells (hiPSCs).
[0017] Preferably, the population of cytotoxic immune cells used in
the compositions and methods of the invention comprises NK cells.
Preferably, the population of cytotoxic immune cells used in the
compositions and methods of the invention comprises NK cells and
.gamma..delta. T-cells. Preferably the population of cytotoxic
immune cells comprises about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35% ,
40%, 45%, 50%, or more of NK cells. Preferably the population of
cytotoxic immune cells comprises about 5% to about 25% of NK cells.
Preferably the population of cytotoxic immune cells used in the
compositions and methods of the invention comprise NK cells,
.gamma..delta. T-cells, and other immunocompetent cells including,
but not limited to: monocytes, dendrites and macrophages.
Preferably, the population of cytotoxic immune cells used in the
compositions and methods of the invention comprise NK cells derived
from human induced pluripotent stem cells (hiPSCs).
[0018] Preferably, the step of obtaining a population of cytotoxic
immune cells genetically modified to be resistant to a therapeutic
agent comprises: obtaining from a subject such as a human subject
or animal subject a population of cytotoxic immune cells, for
example by obtaining a biological sample from the subject including
but not limited to a blood or tissue sample including a tumor
biopsy. The sample may optionally be enriched for cytotoxic immune
cells and other immunocompetent cells and/or the cells present in
the sample may optionally be expanded to increase the population of
the cells present in the sample. The cytotoxic immune cells are
preferably stably transformed or gene edited with a vector
comprising a heterologous nucleic acid sequence operably linked to
a promoter, wherein the heterologous nucleic acid sequence encodes
a polypeptide conferring to the cell resistance to one or more
chemotherapeutic agents.
[0019] Preferably, the invention provides systems for treating a
cancer in a patient comprising a cytotoxic therapeutic agent having
the characteristics of inhibiting the survival of a cancer cell, a
population of cytotoxic immune cells comprising .gamma..delta.
T-cells, NK cells, or any combination thereof and optionally
further comprising other immunocompetent cells and wherein the
cytotoxic immune cells are genetically modified to be resistant to
the cytotoxic therapeutic agent, and an immune checkpoint inhibitor
which blocks cell-surface proteins on cancer cells.
[0020] Preferably, the invention provides systems for treating a
glioblastoma in a patient comprising a therapeutic agent having the
characteristics of inhibiting the survival of a cancer cell and
inducing a stress protein in the cancer cell, a population of
cytotoxic immune cells, wherein said cytotoxic immune cells
comprise .gamma..delta. T-cells, NK cells, or any combination
thereof and optionally further comprise other immunocompetent cells
and wherein said cytotoxic immune cells have been genetically
modified to be resistant to the therapeutic agent, in combination
with an immune checkpoint inhibitor which blocks cell-surface
proteins on cancer cells.
[0021] Preferably, the population of cytotoxic immune cells used in
the compositions and methods of the invention comprise
.gamma..delta. T-cells wherein greater than about 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70% 80%, 90%, or 95% of the .gamma..delta.
T-cells express a polypeptide that confers resistance to a
chemotherapy agent, or isolated compositions comprising
.gamma..delta. T-cells wherein greater than about 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70% 80%, 90%, or 95% of the .gamma..delta.
T-cells comprise a nucleic acid that encodes a polypeptide that
confers resistance to a chemotherapy agent, or isolated
compositions consisting essentially of .gamma..delta. T-cells
comprising a nucleic acid that encodes a polypeptide that confers
resistance to a chemotherapy agent. Preferably, the polypeptide
that confers resistance to a chemotherapy agent is O.sup.6
methylguanine DNA methyltransferase (MGMT), a drug resistant
variant of dihydrofolate reductase (L22Y-DHFR), thymidylate
synthase, and/or multiple drug resistance-1 protein (MDR1).
[0022] Preferably, the population of cytotoxic immune cells used in
the compositions and methods of the invention comprises NK cells
wherein greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%
80%, 90%, or 95% of the NK cells express a polypeptide that confers
resistance to a chemotherapy agent. Preferably, the cytotoxic
immune cells used in compositions and methods of the invention
comprise NK cells where greater than about 50% of the NK cells
express a polypeptide that confers resistance to a chemotherapy
agent. Preferably, the population of cytotoxic immune cells used in
the compositions and methods of the invention comprises wherein
greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% 80%, 90%,
or 95% of the NK cells comprise a nucleic acid that encodes a
polypeptide that confers resistance to a chemotherapy agent.
Preferably, the cytotoxic immune cells used in compositions and
methods of the invention comprise NK cells where greater than about
50% of the NK cells comprise a nucleic acid that encodes a
polypeptide that confers resistance to a chemotherapy agent.
Preferably, the polypeptide expressed by NK cells that confers
resistance to a chemotherapy agent is O.sup.6 methylguanine DNA
methyltransferase (MGMT), a drug resistant variant of dihydrofolate
reductase (L22Y-DHFR), thymidylate synthase, and/or multiple drug
resistance-1 protein (MDR1).
[0023] Preferably, the invention provides compositions comprising
at least one checkpoint inhibitor, and an isolated population of
cytotoxic immune cells comprising .gamma..delta. T-cells NK cells,
or any combination thereof, wherein greater than about 5%, and
preferably greater than about 50% of the population of cytotoxic
immune cells express a polypeptide that confers resistance to a
therapeutic agent capable of inhibiting the survival of a cancer
cell.
BRIEF DESCRIPTION OF THE FIGURES
[0024] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
[0025] FIG. 1 Shows checkpoint molecules expression on
.gamma..delta. T cells, before in vitro culture of PBMCs from Donor
1.
[0026] FIG. 2 Shows upregulation of checkpoint molecules on in
vitro enriched .gamma..delta. T cells from Donor 1.
[0027] FIG. 3 Shows checkpoint molecules expression on
.gamma..delta. T cells, before in vitro culture of PBMCs from Donor
2.
[0028] FIG. 4 Shows upregulation of checkpoint molecules on in
vitro enriched .gamma..delta. T cells from Donor 2.
[0029] FIG. 5 Shows upregulation of PD-1 and CTLA-4 on human
.gamma..delta. T cells upon stimulation with IL2 and Zol.
[0030] FIG. 6 Shows PD-L1expression on glioblastoma tumor
cells.
[0031] FIG. 7 Shows cytotoxicity of GMP manufactured .gamma..delta.
T cells.
DETAILED DESCRIPTION OF THE INVENTION
[0032] This invention is not limited to particular embodiments
described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims.
[0033] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
(unless the context clearly dictates otherwise), between the upper
and lower limit of that range, and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
[0035] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its invention prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided could be
different from the actual publication dates that may need to be
independently confirmed.
[0036] Unless otherwise indicated, the present specification
describes techniques of chemistry, synthetic organic chemistry,
biochemistry, biology, molecular biology, molecular imaging, and
the like, which are within the skill of the art. Such techniques
are explained fully in the literature.
[0037] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete invention and
description of how to perform the methods and use the compositions
and compounds disclosed and claimed herein.
[0038] Unless otherwise indicated, the present invention is not
limited to particular materials, reagents, reaction materials,
manufacturing processes, or the like, as such can vary. It is also
to be understood that the terminology used herein is for purposes
of describing particular features only, and is not intended to be
limiting. It is also possible in the present invention that steps
can be executed in different sequence where this is logically
possible.
[0039] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a support" includes a plurality of
supports. In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings unless a contrary intention is
apparent.
Definitions
[0040] In describing and claiming the disclosed subject matter, the
following terminology will be used in accordance with the
definitions set forth below.
[0041] By "administration" is meant introducing a compound,
biological materials including a cell population, or a combination
thereof, of the present invention into a human or animal subject.
One preferred route of administration of the compounds is
intravenous. Other preferred routes of administration of the
compounds may be intraperitoneal or intrapleural, or via a catheter
to the brain. However, any route of administration, such as oral,
topical, subcutaneous, peritoneal, intra-arterial, inhalation,
vaginal, rectal, nasal, introduction into the cerebrospinal fluid,
or instillation into body compartments can be used. Direct
injection into a target tissue site such as a solid tumor is also
contemplated.
[0042] The term "therapeutic agent" as used herein refers to a
compound or a derivative thereof that can interact with a cancer
cell, thereby reducing the proliferative status of the cell and/or
killing the cell. Examples of therapeutic agents include, but are
not limited to, chemotherapeutic agents which include, but are not
limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide);
metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil or
derivatives thereof); a substituted nucleotide; a substituted
nucleoside; DNA demethylating agents (also known as
antimetabolites; e.g., azacitidine); antitumor antibiotics (e.g.,
mitomycin, adriamycin);
[0043] plant-derived antitumor agents (e.g., vincristine,
vindesine, TAXOL.RTM., paclitaxel, abraxane); cisplatin;
carboplatin; etoposide; and the like. Such agents may further
include, but are not limited to, the anti-cancer agents
trimethotrexate (TMTX); temozolomide; raltitrexed;
S-(4-Nitrobenzyl)-6-thioinosine (NBMPR); 6-benzyguanidine (6-BG);
nitrosoureas [e.g., bis-chloronitrosourea (BCNU; carmustine),
lomustine (CCNU) +/-Procarbazine and Vincristine (PCV regimen),
fotemustine]; cytarabine; and camptothecin; or a therapeutic
derivative of any thereof.
[0044] The term "therapeutically effective amount" as used herein
refers to that amount of the compound or therapeutically active
composition being administered that will relieve to some extent one
or more of the symptoms of a disease, a condition, or a disorder
being treated. In reference to cancer or pathologies related to
unregulated cell division, a therapeutically effective amount
refers to that amount which has the effect of (1) reducing the size
of a tumor, (2) inhibiting (that is, slowing to some extent,
preferably stopping) aberrant cell division, for example cancer
cell division, (3) preventing or reducing the metastasis of cancer
cells, and/or, (4) relieving to some extent (or, preferably,
eliminating) one or more symptoms associated with a pathology
related to or caused in part by unregulated or aberrant cellular
division, including for example, cancer, or angiogenesis.
[0045] The terms "treating" or "treatment" of a disease (or a
condition or a disorder) as used herein refer to preventing the
disease from occurring in a human subject or an animal subject that
may be predisposed to the disease but does not yet experience or
exhibit symptoms of the disease (prophylactic treatment),
inhibiting the disease (slowing or arresting its development),
providing relief from the symptoms or side-effects of the disease
(including palliative treatment), and causing regression of the
disease. With regard to cancer, these terms also mean that the life
expectancy of an individual affected with a cancer may be increased
or that one or more of the symptoms of the disease will be reduced.
With regard to cancer, "treating" also includes enhancing or
prolonging an anti-tumor response in a subject.
[0046] As used herein any form of administration of a
"combination", "combined therapy" and/or "combined treatment
regimen" refers to at least two therapeutically active drugs or
compositions which may be administered simultaneously, in either
separate or combined formulations, or sequentially at different
times separated by minutes, hours or days, but in some way act
together to provide the desired therapeutic response.
[0047] The term "enhancing", as used herein, refers to allowing a
subject or tumor cell to improve its ability to respond to a
treatment disclosed herein. For example, an enhanced response may
comprise an increase in responsiveness of at least 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 98% or more. As used herein, "enhancing" can also
refer to enhancing the number of subjects who respond to a
treatment such as a combination therapy comprising chemotherapy,
drug-resistant immunocompetent cells, and immune checkpoint
inhibitors. For example, an enhanced response may refer to a total
percentage of subjects who respond to a treatment wherein the
percentage is of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or
more.
[0048] The terms "subject" and "patient" as used herein include
humans, mammals (e.g., cats, dogs, horses, etc.), living cells, and
other living organisms. A living organism can be as simple as, for
example, a single eukaryotic cell or as complex as a mammal.
Typical patients are mammals, particularly primates, especially
humans. For veterinary applications, a wide variety of subjects
will be suitable, e.g., livestock such as cattle, sheep, goats,
cows, swine, and the like; poultry such as chickens, ducks, geese,
turkeys, and the like; and domesticated animals particularly pets
such as dogs and cats. For diagnostic or research applications, a
wide variety of mammals will be suitable subjects, including
rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine
such as inbred pigs and the like. Preferably, a system includes a
sample and a subject. The term "living host" refers to host or
organisms noted above that are alive and are not dead. The term
"living host" refers to the entire host or organism and not just a
part excised (e.g., a liver or other organ) from the living
host.
[0049] The term ".gamma..delta. T-cells (gamma delta T-cells)" as
used herein refers to a small subset of T-cells that express a
distinct T-cell receptor (TCR) on their surface. A majority of
T-cells have a TCR composed of two glycoprotein chains called
.alpha.- and .beta.-TCR chains. In contrast, in .gamma..delta.
T-cells, the TCR is made up of one .gamma.-chain and one
.delta.-chain. This group of T-cells is usually much less common
than .alpha..beta. T-cells, but are found at their highest
abundance in the gut mucosa, within a population of lymphocytes
known as intraepithelial lymphocytes (IELs). The antigenic
molecules that activate .gamma..delta. T-cells are still largely
unknown. However, .gamma..delta. T-cells are peculiar in that they
do not seem to require antigen processing and MHC presentation of
peptide epitopes although some recognize MHC class D3 molecules.
Furthermore, .gamma..delta. T-cells are believed to have a
prominent role in recognition of lipid antigens, and to respond to
stress-related antigens such as, MIC-A and MIC-B.
[0050] The term "human induced pluripotent stem cells" (hiPSCs) as
used herein refers to a type of pluripotent stem cells that can be
generated directly from human adult cells such as skin or blood
cells that have been reprogrammed back into an embryonic-like
pluripotent state that enables the generation of any other type of
human cell, for example a therapeutic immunocompetent cell. The
human adult cells from which the hiPSCs may be obtained from the
patient to be treated or the adult cells may be obtained from a
different individual. Human adult cells may be transformed into
pluripotent stem cells with, for example, a retroviral system or a
lentiviral system for introducing genes encoding transcription
factors that are able to convert adult cells into pluripotent stem
cells.
[0051] The term "antibody", as used herein, refers to an
immunoglobulin or a part thereof, and encompasses any polypeptide
comprising an antigen-binding site regardless of the source,
species of origin, method of production, and characteristics.
Antibodies may be comprised of heavy and/or light chains or
fragments thereof. Antibodies or antigen-binding fragments,
variants, or derivatives thereof of the invention include, but are
not limited to, polyclonal, monoclonal, multispecific, human,
humanized, primatized, or chimeric antibodies, single chain
antibodies, epitope-binding fragments, e.g., Fab, Fab' and
F(ab').sub.2, Fd, Fvs, single-chain Fvs (scFv), single-chain
antibodies, disulfide-linked Fvs (sdFv), fragments comprising
either a VL or VH domain, fragments produced by a Fab expression
library, and anti-idiotypic (anti-Id) antibodies. ScFv molecules
are known in the art and are described, e.g., in U.S. Pat. No.
5,892,019. Immunoglobulin or antibody molecules of the invention
can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class
(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of
immunoglobulin molecule.
[0052] The term "biologic therapeutic" or "biopharmaceutical", as
used herein, refers to any medicinal product manufactured in or
extracted from biological sources. Biopharmaceuticals are distinct
from chemically synthesized pharmaceutical products. Examples of
biopharmaceuticals include vaccines, blood or blood components,
allergenics, somatic cells, gene therapies, tissues, recombinant
therapeutic proteins, including antibody therapeutics and fusion
proteins, and living cells. Biologics can be composed of sugars,
proteins or nucleic acids or complex combinations of these
substances, or may be living entities such as cells and tissues.
Biologics are isolated from a variety of natural sources human,
animal or microorganism and may be produced by biotechnology
methods and other technologies. Specific examples of biologic
therapeutics include, but are not limited to, immunostimulatory
agents, T cell growth factors, interleukins, antibodies, fusion
proteins and vaccines, such as cancer vaccines.
[0053] The term "cancer", as used herein, shall be given its
ordinary meaning, as a general term for diseases in which abnormal
cells divide without control. In particular, and in the context of
the embodiments of the present invention, cancer refers to
angiogenesis-related cancer. Cancer cells can invade nearby tissues
and can spread through the bloodstream and lymphatic system to
other parts of the body. There are several main types of cancer,
for example, carcinoma is cancer that begins in the skin or in
tissues that line or cover internal organs. Sarcoma is cancer that
begins in bone, cartilage, fat, muscle, blood vessels, or other
connective or supportive tissue. Leukemia is cancer that starts in
blood-forming tissue such as the bone marrow, and causes large
numbers of abnormal blood cells to be produced and enter the
bloodstream. Lymphoma is cancer that begins in the cells of the
immune system.
[0054] When normal cells lose their ability to behave as a
specified, controlled and coordinated unit, a tumor is formed.
Generally, a solid tumor is an abnormal mass of tissue that usually
does not contain cysts or liquid areas (some brain tumors do have
cysts and central necrotic areas filled with liquid). A single
tumor may even have different populations of cells within it, with
differing processes that have gone awry. Solid tumors may be benign
(not cancerous), or malignant (cancerous). Different types of solid
tumors are named for the type of cells that form them. Examples of
solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias
(cancers of the blood) generally do not form solid tumors.
[0055] Representative cancers include, but are not limited to,
Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia,
Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma;
Adrenocortical Carcinoma, Childhood; AIDS-Related Lymphoma;
AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood
Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer,
Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone
Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Glioblastoma,
Childhood; Glioblastoma, Adult; Brain Stem Glioma, Childhood; Brain
Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain
Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral
Astrocytoma/Malignant Glioma, Childhood; Brain Tumor, Ependymoma,
Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor,
Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain
Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain
Tumor, Childhood (Other); Breast Cancer; Breast Cancer and
Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial
Adenomas/Carcinoids, Childhood: Carcinoid Tumor, Childhood;
Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical;
Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central
Nervous System Lymphoma, Primary; Cerebellar Astrocytoma,
Childhood; Cerebral Astrocytoma/Malignant Glioma, Childhood;
Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia;
Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders;
Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal
Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer;
Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal
Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors;
Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell
Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular
Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric
(Stomach) Cancer; Gastric (Stomach) Cancer, Childhood;
Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial,
Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian;
Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem;
Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell
Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer,
Adult (Primary); Hepatocellular (Liver) Cancer, Childhood
(Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma,
Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal
Cancer; Hypothalamic and Visual Pathway Glioma, Childhood;
Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas);
Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal
Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia,
Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult;
Leukemia,
[0056] Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic;
Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral
Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer,
Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer,
Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic
Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma,
AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma,
Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's;
Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma,
Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma,
Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous
System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer;
Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood;
Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma,
Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant;
Metastatic Squamous Neck Cancer with Occult Primary; Multiple
Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma
Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes;
Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute;
Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal
Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer;
Nasopharyngeal Cancer, Childhood; Neuroblastoma; Neurofibroma;
Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood;
Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung
Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer;
Oropharyngeal Cancer; Osteosarcoma/Malignant Fibrous Histiocytoma
of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer;
Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor;
Pancreatic Cancer; Pancreatic Cancer, Childhood', Pancreatic
Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer;
Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and
Supratentorial Primitive Neuroectodermal Tumors, Childhood;
Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma;
Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy
and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma;
Primary Central Nervous System Lymphoma; Primary Liver Cancer,
Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal
Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood;
Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma;
Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland'
Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma,
Kaposi's; Sarcoma (Osteosarcoma)/Malignant Fibrous Histiocytoma of
Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue,
Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin
Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin
Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine
Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood;
Squamous Neck Cancer with Occult Primary, Metastatic; Stomach
(Gastric) Cancer; Stomach (Gastric) Cancer, Childhood;
Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell
Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood;
Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood;
Transitional Cell Cancer of the Renal Pelvis and Ureter;
Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of,
Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis,
Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal
Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar
Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor, among
others.
[0057] A tumor can be classified as malignant or benign. In both
cases, there is an abnormal aggregation and proliferation of cells.
In the case of a malignant tumor, these cells behave more
aggressively, acquiring properties of increased invasiveness.
Ultimately, the tumor cells may even gain the ability to break away
from the microscopic environment in which they originated, spread
to another area of the body (with a very different environment, not
normally conducive to their growth), and continue their rapid
growth and division in this new location. This is called
metastasis. Once malignant cells have metastasized, achieving a
cure is more difficult. Benign tumors have less of a tendency to
invade and are less likely to metastasize.
[0058] Brain tumors spread extensively within the brain but do not
usually metastasize outside the brain. Gliomas are very invasive
inside the brain, even crossing hemispheres. They do divide in an
uncontrolled manner, though. Depending on their location, they can
be just as life threatening as malignant lesions. An example of
this would be a benign tumor in the brain, which can grow and
occupy space within the skull, leading to increased pressure on the
brain.
[0059] The term "enriched", as used herein, refers to increasing
the total percentage of one or more cytotoxic immune cell types
present (e.g., .gamma..delta. T-cells and/or NK cells)1 in a
sample, relative to the total percentage of the same one or more
cell types prior to enrichment, as disclosed herein. For example, a
sample that is "enriched" for a for one or more types of cytotoxic
immune cell may comprise between about 10% to 100% of the one or
more cytotoxic immune cell types in the sample, whereas the total
percentage of one or more of the cytotoxic immune cell types in a
sample prior to enrichment was, for example, between 0% and 10%.
Preferably, an enriched sample comprises at least 10%, 15%, 20%,
25%, 30%, 35%, 40%, 50%, 60% ,70%, 80%, 90% or 100%, of one or more
types of cytotoxic immune cell. Samples may be enriched for one or
more cell types using standard techniques, for example, flow
cytometry techniques.
[0060] The term "highly enriched", as used herein, refers to
increasing the total percentage of one or more cytotoxic immune
cell types in a sample such that the one or more cytotoxic immune
cell types may comprise between at least about 70% to about 100% of
the cytotoxic immune cell type in the sample, whereas the total
percentage of that same type of cytotoxic immune cell prior to
enrichment was, for example, between 0% and 10%. Preferably, a
highly enriched sample comprises at least 70%, 75%, 80%, 85%, 90%,
95%, 99% or more of one or more types of cytotoxic immune cell.
Samples may be highly enriched for one or more cell types using
standard techniques, for example, flow cytometry techniques.
[0061] The terms "expanded" and "expansion" as used herein with
regard to expansion of one or more cytotoxic immune cells in a
sample means to increase in the number of one or more cytotoxic
immune cells in a sample by, for example about at least 2-fold,
preferably by about 5-fold, preferably by at least 10-fold,
preferably about at least 50-fold or more. Expansion of a cytotoxic
immune cell population can be accomplished by any number of methods
as are known in the art. For example, T-cells can be rapidly
expanded using non-specific T-cells receptor stimulation in the
presence of feeder lymphocytes and either interleukin-2 (IL-2) or
interleukin-15 ( IL-15), with IL-2 being preferred. The
non-specific T-cells receptor stimulus can include around 30 ng/ml
of OKT3, a mouse monoclonal anti-CD3 antibody (available from
ORTHO-MCNEIL.RTM., Raritan, N.J.). Alternatively T-cells can be
rapidly expanded by stimulation of peripheral blood mononuclear
cells (PBMC) in vitro with one or more antigens (including
antigenic portions thereof, such as epitope(s), or a cell) of the
cancer, which can be optionally expressed from a vector, such as an
human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3
.mu.M MART-1:26-35 (27 L) or gp100:209-217 (210M), in the presence
of a T-cell growth factor, such as 300 IU/ml IL-2 or IL-15, with
IL-2 being preferred.
[0062] The term "fusion protein", as used herein, refers to
chimeric molecules, which comprise, for example, an immunoglobulin
antigen-binding domain with at least one target binding site, and
at least one heterologous portion, i.e., a portion with which it is
not naturally linked in nature. The amino acid sequences may
normally exist in separate proteins that are brought together in
the fusion polypeptide or they may normally exist in the same
protein but are placed in a new arrangement in the fusion
polypeptide. Fusion proteins may be created, for example, by
chemical synthesis, or by creating and translating a polynucleotide
in which the peptide regions are encoded in the desired
relationship.
[0063] The term "reducing a cancer" as used herein refers to a
reduction in the size or volume of a tumor mass, a decrease in the
number of metastasized tumors in a subject, a decrease in the
proliferative status (the degree to which the cancer cells are
multiplying) of the cancer cells, and the like.
[0064] The terms "isolated' and isolated population of cells" as
used herein refers to a cell or a plurality of cells removed from
the tissue or state in which they are found in a subject. The terms
may further include cells that have been separated according to
such parameters as, but not limited to, cell surface markers, a
reporter marker such as a dye or label.
[0065] The term "expressed" or "expression" as used herein refers
to the transcription from a gene to give an RNA nucleic acid
molecule at least complementary in part to a region of one of the
two nucleic acid strands of the gene. The term "expressed" or
"expression" as used herein also refers to the translation from
said RNA nucleic acid molecule to give a protein, a polypeptide, or
a portion or fragment thereof.
[0066] The term "promoter" as used herein refers to the DNA
sequence that determines the site of transcription initiation from
an RNA polymerase. A "promoter-proximal element" may be a
regulatory sequence within about 200 base pairs of the
transcription start site.
[0067] The term "recombinant cell" refers to a cell that has a new
combination of nucleic acid segments that are not covalently linked
to each other in nature. A new combination of nucleic acid segments
can be introduced into an organism using a wide array of nucleic
acid manipulation techniques available to those skilled in the art.
A recombinant cell can be a single eukaryotic cell, or a single
prokaryotic cell, or a mammalian cell. The recombinant cell may
harbor a vector that is extragenomic. An extragenomic nucleic acid
vector does not insert into the cell's genome. A recombinant cell
may further harbor a vector or a portion thereof that is
intragenomic. The term "intragenomic" defines a nucleic acid
construct incorporated within the recombinant cell's genome.
[0068] The terms "recombinant nucleic acid" and "recombinant DNA"
as used herein refer to combinations of at least two nucleic acid
sequences that are not naturally found in a eukaryotic or
prokaryotic cell. The nucleic acid sequences include, but are not
limited to, nucleic acid vectors, gene expression regulatory
elements, origins of replication, suitable gene sequences that when
expressed confer antibiotic resistance, protein-encoding sequences,
and the like. The term "recombinant polypeptide" is meant to
include a polypeptide produced by recombinant DNA techniques such
that it is distinct from a naturally occurring polypeptide either
in its location, purity or structure. Generally, such a recombinant
polypeptide will be present in a cell in an amount different from
that normally observed in nature.
[0069] The terms "operably" or "operatively linked" as used herein
refer to the configuration of the coding and control sequences so
as to perform the desired function. Thus, control sequences
operably linked to a coding sequence are capable of effecting the
expression of the coding sequence. A coding sequence is operably
linked to or under the control of transcriptional regulatory
regions in a cell when DNA polymerase will bind the promoter
sequence and transcribe the coding sequence into mRNA that can be
translated into the encoded protein. The control sequences need not
be contiguous with the coding sequence, so long as they function to
direct the expression thereof. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding
sequence.
[0070] The terms "heterologous" and "exogenous" as they relate to
nucleic acid sequences such as coding sequences and control
sequences denote sequences that are not normally associated with a
region of a recombinant construct or with a particular chromosomal
locus, and/or are not normally associated with a particular cell.
Thus, a "heterologous" region of a nucleic acid construct is an
identifiable segment of nucleic acid within or attached to another
nucleic acid molecule that is not found in association with the
other molecule in nature. For example, a heterologous region of a
construct tip could include a coding sequence flanked by sequences
not found in association with the coding sequence in nature.
Another example of a heterologous coding sequence is a construct
where the coding sequence itself is not found in nature (e.g.,
synthetic sequences having codons different from the native gene).
Similarly, a host cell transformed with a construct, which is not
normally present in the host cell, would be considered heterologous
for purposes of this invention.
[0071] Preferably, the promoter will be modified by the addition or
deletion of sequences, or replaced with alternative sequences,
including natural and synthetic sequences as well as sequences that
may be a combination of synthetic and natural sequences. Many
eukaryotic promoters contain two types of recognition sequences:
the TATA box and the upstream promoter elements. The former,
located upstream of the transcription initiation site, is involved
in directing RNA polymerase to initiate transcription at the
correct site, while the latter appears to determine the rate of
transcription and is upstream of the TATA box. Enhancer elements
can also stimulate transcription from linked promoters, but many
function exclusively in a particular cell type. Many
enhancer/promoter elements derived from viruses, e.g., the SV40,
the Rous sarcoma virus (RSV), and CMV promoters are active in a
wide array of cell types, and are termed "constitutive" or
"ubiquitous." The nucleic acid sequence inserted in the cloning
site may have any open reading frame encoding a polypeptide of
interest, with the proviso that where the coding sequence encodes a
polypeptide of interest, it should lack cryptic splice sites that
can block production of appropriate mRNA molecules and/or produce
aberrantly spliced or abnormal mRNA molecules.
[0072] The termination region that is employed primarily will be
one of convenience, since termination regions appear to be
relatively interchangeable. The termination region may be native to
the intended nucleic acid sequence of interest, or may be derived
from another source.
[0073] The term "targeted therapy", as used herein, refers to any
therapeutic molecule that targets any aspect of the immune
system.
[0074] The term "vector" as used herein refers to a polynucleotide
comprised of single strand, double strand, circular, or supercoiled
DNA or RNA. A typical vector may be comprised of the following
elements operatively linked at appropriate distances for allowing
functional gene expression: replication origin, promoter, enhancer,
5' mRNA leader sequence, ribosomal binding site, nucleic acid
cassette, termination and polyadenylation sites, and selectable
marker sequences. One or more of these elements may be omitted in
specific applications. The nucleic acid cassette can include a
restriction site for insertion of the nucleic acid sequence to be
expressed. In a functional vector the nucleic acid cassette
contains the nucleic acid sequence to be expressed including
translation initiation and termination sites.
[0075] A vector is constructed so that the particular coding
sequence is located in the vector with the appropriate regulatory
sequences, the positioning and orientation of the coding sequence
with respect to the control sequences being such that the coding
sequence is transcribed under the "control" of the control or
regulatory sequences. Modification of the sequences encoding the
particular protein of interest may be desirable to achieve this
end. For example, in some cases it may be necessary to modify the
sequence so that it may be attached to the control sequences with
the appropriate orientation; or to maintain the reading frame. The
control sequences and other regulatory sequences may be ligated to
the coding sequence prior to insertion into a vector.
Alternatively, the coding sequence can be cloned directly into an
expression vector that already contains the control sequences and
an appropriate restriction site that is in reading frame with and
under regulatory control of the control sequences.
[0076] The term "lentiviral-based vector" as used herein refers to
a lentiviral vector designed to operably insert an exogenous
polynucleotide sequence into a host genome in a site-specific
manner. Lentiviral-based targeting vectors may be based on, but is
not limited to, for example, HIV-1, HIV-2, simian immunodeficiency
virus (SIV), or feline immunodeficiency virus (Hy). Preferably, the
lentiviral-based targeting vector is an HIV-based targeting vector.
This vector may comprise all or a portion of the polynucleotide
sequence of HIV.
[0077] The terms "transformation", "transduction" and
"transduction" all denote the introduction of a polynucleotide into
a recipient cell or cells.
Combination Drug Resistant Immunotherapy with Immune Checkpoint
Inhibitors
[0078] A major limitation to chemotherapy treatments for cancer is
drug induced immune toxicity which causes the killing of
immunocompetent cells and loss of an effective immune system that
would otherwise ward off undesirable infections or provide a
defense against cancer cells. One strategy to combat the severe
toxic effects of chemotherapy would be to selectively genetically
modify cytotoxic immunocompetent cells by the introduction of
retroviral vectors designed to express cDNA sequences that confer
drug resistance, which can actively target those cancer cells able
to resist the simultaneous administration of a chemotherapeutic
agent.
[0079] Immune checkpoint proteins regulate T cell function in the
immune system. T cells play a central role in cell-mediated
immunity. Checkpoint proteins interact with specific ligands that
send a signal into the T cell and essentially switch off or inhibit
T cell function. Cancer cells take advantage of this system by
driving high levels of expression of checkpoint proteins on their
surface that results in control of the T cells expressing
checkpoint proteins on the surface of T cells that enter the tumor
microenvironment, thus suppressing the anticancer immune response.
As such, inhibition of checkpoint proteins would result in
restoration of T cell function and an immune response to the cancer
cells. Examples of checkpoint proteins include, but are not limited
to CTLA-4, PDL1 (B7-H1, CD274), PDL2 (B7-DC, CD273), PD1, B7-H3
(CD276), B7-H4 (B7-S1, B7x, VCTN1), BTLA (CD272), HVEM, TIM3
(HAVcr2), GAL9, LAG3 (CD223), VISTA, KIR, 2B4 (CD244; belongs to
the CD2 family of molecules and is expressed on all NK,
.gamma..delta., and memory CD8.sup.+ (.alpha..beta.) T cells),
CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2
kinases, OX40, A2aR and various B-7 family ligands.
[0080] Programmed cell death protein 1 (PD-1) is a 288 amino acid
cell surface protein molecule that is expressed on T cells and
pro-B cells and plays a role in their fate/differentiation. PD-1
has two ligands, PD-L1 and PD-L2, which are members of the B7
family. PD-L1 protein is upregulated on macrophages and dendritic
cells (DC) in response to LPS and GM-CSF treatment, and on T cells
and B cells upon TCR and B cell receptor signaling. PD-1 negatively
regulates T cell responses.
[0081] PD-1 plays a role in tumor-specific escape from immune
surveillance. It has been demonstrated that PD-1 is highly
expressed in tumor-specific cytotoxic T lymphocytes (CTLs) in both
chronic myelogenous leukemia (CIVIL) and acute myelogenous leukemia
(AML). PD-1 is also up-regulated in melanoma infiltrating T
lymphocytes (TILs) [Dotti (2009) Blood 114 (8): 1457-58]. Tumors
have been found to express the PD-1 ligands PDL-1 and PDL-2. For
example, PDL-1 is found in glioblastoma, especially in mesenchymal
subtype. (Nat Rev Neurol 2015: 11:504-515). The expression of PDL-1
and PDL-2 by tumors, combined with the up-regulation of PD-1 in
CTLs, may be a contributory factor in the loss in T cell
functionality and the inability of CTLs to mediate an effective
anti-tumor response. Researchers have shown that in mice
chronically infected with lymphocytic choriomeningitis virus
(LCMV), administration of anti-PD-1 antibodies blocked PD-1-PDL
interaction and was able to restore some T cell functionality
(proliferation and cytokine secretion), and lead to a decrease in
viral load (Barber et al (2006) Nature 439 (9): 682-687).
[0082] Tumor cells themselves expresses PD-L1 and are thought to
limit T cell responses via this mechanism. It has further been
shown that inhibition of PD-1 results in expansion of effector T
cells and restriction of T regulatory cell population in B16
melanoma models. Blockade of PD-1, CTLA-4, or IDO (indoleamine
2,3-dioxygenase) restores IL-2 production and allows for increased
proliferation of CD8+ T cells present in the tumor
microenvironment. It has been shown that anti-PDL1 treatment
rescues and allows expansion of antigen-specific vaccine-generated
CD8+ T cells to reject tumor. These data suggest that PD-1/PD-L1
axis regulates activated tumor-specific T cells.
[0083] Clinical trials in melanoma, non-small cell lung cancer, and
renal cell carcinoma have shown anti-tumor responses in some
patients with anti-PD-1 blockade. Significant benefits with PD-1
inhibition in cases of advanced melanoma, non-small-cell lung,
prostate, renal-cell, bladder, and colorectal cancer have also been
described. Studies in murine models have applied this evidence to
glioma therapy. A decrease in tumor-infiltrating Tregs and
increased survival when combinatorial treatment of IDO, CTLA-4, and
PD-L1 inhibitors was administered has been described.
[0084] There are several PD-1 inhibitors currently being tested in
clinical trials. CT-011 is a humanized IgG1 monoclonal antibody
against PD-1. A phase II clinical trial in subjects with diffuse
large B-cell lymphoma (DLBCL) who have undergone autologous stem
cell transplantation was recently completed. Preliminary results
demonstrated that 70% of subjects were progression-free at the end
of the follow-up period, compared with 47% in the control group,
and 82% of subjects were alive, compared with 62% in the control
group. This trial determined that CT-011 not only blocks PD-1
function, but it also augments the activity of natural killer
cells, thus intensifying the antitumor immune response.
[0085] BMS 936558 is a fully human IgG4 monoclonal antibody
targeting PD-1 agents. In a phase I trial, biweekly administration
of BMS-936558 in subjects with advanced, treatment-refractory
malignancies showed durable partial or complete regressions. The
most significant response rate was observed in subjects with
melanoma (28%) and renal cell carcinoma (27%), but substantial
clinical activity was also observed in subjects with non-small cell
lung cancer (NSCLC), and some responses persisted for more than a
year. It was also relatively well tolerated; grade.gtoreq.3 adverse
events occurred in 14% of subjects.
[0086] BMS 936559 is a fully human IgG4 monoclonal antibody that
targets the PD-1 ligand PD-L1. Phase I results showed that biweekly
administration of this drug led to durable responses, especially in
subjects with melanoma. Objective response rates ranged from 6% to
17% depending on the cancer type in subjects with advanced-stage
NSCLC, melanoma, RCC, or ovarian cancer, with some subjects
experiencing responses lasting a year or longer.
[0087] MK 3475 is a humanized IgG4 anti-PD-1 monoclonal antibody in
phase I development in a five-part study evaluating the dosing,
safety, and tolerability of the drug in subjects with progressive,
locally advanced, or metastatic carcinoma, melanoma, or NSCLC.
[0088] MPDL 3280A is a monoclonal antibody, which also targets
PD-L1, undergoing phase I testing in combination with the BRAF
inhibitor vemurafenib in subjects with BRAF V600-mutant metastatic
melanoma and in combination with bevacizumab, which targets
vascular endothelial growth factor receptor (VEGFR), with or
without chemotherapy in subjects with advanced solid tumors.
[0089] AMP 224 is a fusion protein of the extracellular domain of
the second PD-1 ligand, PD-L2, and IgG1, which has the potential to
block the PD-L2/PD-1 interaction. AMP-224 is currently undergoing
phase I testing as monotherapy in subjects with advanced
cancer.
[0090] Medi 4736 is an anti-PD-L1 antibody in phase I clinical
testing in subjects with advanced malignant melanoma, renal cell
carcinoma, NSCLC, and colorectal cancer.
[0091] CTLA4 (cytotoxic T-lymphocyte-associated protein), is a
protein receptor that down regulates the immune system. CTLA4 is
found on the surface of T cells, which lead the cellular immune
attack on antigens. The T cell attack can be turned on by
stimulating the CD28 receptor on the T cell. The T cell attack can
be turned off by stimulating the CTLA4 receptor. A first-in-class
immunotherapy, ipilimumab (YERVOY.RTM.), a monoclonal antibody that
targets CTLA-4 on the surface of T cells, was for the treatment of
melanoma.
[0092] Preferably, the present invention provides a method of
treating cancer in a subject in need thereof comprising
administering to the subject a therapeutic agent in combination
with a drug-resistant immunocompetent cell and an immune checkpoint
inhibitor. Preferably, the checkpoint inhibitor is a biologic
therapeutic or a small molecule. Preferably, the checkpoint
inhibitor is a monoclonal antibody, a humanized antibody, a fully
human antibody, a fusion protein or a combination thereof.
Preferably, the checkpoint inhibitor inhibits a checkpoint protein
which may be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM,
TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2,
A2aR, OX40, B-7 family ligands or a combination thereof.
Preferably, the checkpoint inhibitor interacts with a ligand of a
checkpoint protein which may be CTLA-4, PDL1, PDL2, PD1, B7-H3,
B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160,
CGEN-15049, CHK 1, CHK2, OX40, A2aR, B-7 family ligands or a
combination thereof.
[0093] Preferably, the therapeutic agent is a chemotherapeutic
agent, an immunostimulatory agent, a T cell growth factor, an
interleukin, an antibody, a vaccine or any combination thereof.
Preferably, the interleukin is IL-7 or IL-15. Preferably, the
interleukin is glycosylated IL-7.
[0094] Preferably, the drug-resistant immunocompetent cell,
therapeutic agent, and checkpoint inhibitor are administered
simultaneously, or sequentially in any order, either singly, or one
followed by the other two simultaneously, or two simultaneously
followed by the third. Preferably, the drug-resistant
immunocompetent cell is administered prior to the therapeutic agent
and the immune checkpoint inhibitor. Preferably, the checkpoint
inhibitor is a PD-1 inhibitor or a CTLA-4 inhibitor.
[0095] Preferably, the population of cytotoxic immune cells used in
the compositions and methods of the invention comprises
.gamma..delta. T-cells, NK cells, or any combination thereof.
Preferably the population of cytotoxic immune cells comprises about
10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100%
.gamma..delta. T-cells. Preferably the population of cytotoxic
immune cells comprises about 50% to about 95% .gamma..delta.
T-cells. Preferably the population of cytotoxic immune cells used
in the compositions and methods of the invention comprises
.gamma..delta. T-cells and natural killer (NK) cells. Preferably
the population of cytotoxic immune cells comprises about 1%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more of NK cells.
Preferably the population of cytotoxic immune cells comprises about
5% to about 25% of NK cells.
[0096] Preferably, the isolated compositions of cytotoxic immune
cells used in the compositions and methods of the invention
comprise .gamma..delta. T-cells, NK cells or any combination
thereof and optionally further comprise other immunocompetent cells
including but not limited to: monocytes, macrophages and dendritic
cells wherein greater than about 5%, greater than about 10%,
greater than about 20%, greater than about 30%, greater than about
40% greater than about 50% or more of the cytotoxic immune cells
present in the composition express a polypeptide that confers
resistance to a chemotherapy agent. Preferably the isolated
compositions of cytotoxic immune cells used in accordance with the
invention comprises .gamma..delta. T-cells, NK cells or any
combination thereof wherein at least about 50% or more of the
.gamma..delta. T-cells (when present) and greater than about 50% of
the NK cells (when present) express a polypeptide that confers
resistance to a chemotherapy agent. Preferably, the polypeptide
that confers resistance to a chemotherapy agent is O.sup.6
methylguanine DNA methyltransferase (MGMT), a drug resistant
variant of dihydrofolate reductase (L22Y-DHFR), thymidylate
synthase, multiple drug resistance-1 protein (MDR1).
[0097] Preferably, the invention provides methods of treating
cancer in a subject comprising: administering to a subject in need
thereof, an immune checkpoint inhibitor in combination with a
chemotherapy agent and a chemotherapy resistant composition of
immune competent cells comprising .gamma..delta. T-cells, NK cells
or any combination thereof and further optionally comprising other
immunocompetent cells including but not limited to: monocytes,
macrophages and dendritic cells that are genetically engineered to
express a polypeptide that confers resistance to the chemotherapy
agent. The generation and expansion of compositions comprising
drug-resistant .gamma..delta. T-cells, NK cells or any combination
thereof ex vivo can allow, in this setting, for administration of
immunocompetent cell-based therapy concurrently with chemotherapy
and checkpoint inhibitors, potentially improving tumor clearance
while anti-tumor immunity is established and maintained, which
might possibly lead to long-term tumor clearance. Preferably, the
immunocompetent cells are genetically engineered to express at
least one, at least two, at least three or more different tumor
recognition moieties wherein each tumor recognition moiety
recognizes a tumor antigen.
[0098] Preferably, the invention provides methods of treating
cancers, and in particular cancerous tumors. The methods of the
invention combine the use of chemotherapeutic agents that can kill
or reduce the proliferation of cancerous cells, with immunotherapy
and immune checkpoint inhibitors to effectively eliminate those
cancerous cells that develop drug resistance or otherwise escape
the chemotherapeutic agent. The methods of the present invention
provide for isolating cytotoxic immune cells, including, but not
limited to, .gamma..delta. T-cells either from a patient to be
treated or from another source, as described, for example, by Lamb
L. S. in U.S. Pat. No. 7,078,034, incorporated herein by reference
in its entirety. The isolated cells may then be transfected with a
nucleic acid vector comprising a heterologous nucleotide sequence
encoding a polypeptide that confers resistance to a selected
chemotherapeutic agent to the cell. The patient in need of
treatment for a cancer, and in particular a tumor, may then receive
a dose, or doses, of the transfected T-cells before, after or with
the chemotherapeutic agent and the checkpoint inhibitor. The agent
itself, while intended to be toxic to the targeted cancer cells,
and will reduce the proliferation and viability of the cells, may
also induce the microenvironment of the tumor to reduce local
tumor-derived immunosuppression and improve migration of cells into
the tumor as well as formation on the cell surface of the cancer
cells of stress-related proteins. Transfected .gamma..delta.
T-cells, for example, have the characteristic of being able to
recognize and therefore target such stress-related ligands, thereby
specifically or preferentially targeting the cancer cells.
[0099] Preferably, the genetically modified cytotoxic immune cells
comprising .gamma..delta. T-cells, NK cells or combinations
thereof, may be delivered to the targeted tumor directly by such
as, but not limited to, direct injection into the tumor mass,
delivery to a blood vessel entering the tumor mass, or a
combination of both. For example, it is contemplated that the cells
may be delivered to a glioblastoma mass in the brain of a patient
by direct implantation through a cannulated needle or catheter
inserted into the tumor mass, intracavity post-resection,
intraperitoneal or intraventricular.
[0100] Preferably in some protocols that combine immunotherapy and
chemotherapy to treat cancers in a subject human or animal patient,
cytokines (IL-2, IL-12, GM-CSF, and the like) may be delivered to a
patient to boost the formation of cytotoxic lymphocytes. In other
methods, an anti-CD137 specific antibody may be employed. CD137 is
a member of the tumor necrosis factor (TNF)/nerve growth factor
(NGF) family of receptors and is expressed by activated T- and
B-lymphocytes and monocytes; its ligand has been found to play an
important role in the regulation of immune responses. An anti-CD137
monoclonal antibody can specifically bind to CD137-expressing
immune cells such as activated T-cells and freshly isolated mouse
dendritic cells (DCs), thereby stimulating an immune response, in
particular of a cytotoxic T cell response, against tumor cells.
[0101] Preferably, the invention provides methods for treating
cancer in a patient, comprising the steps of: obtaining a
population of cytotoxic immune cells, where the isolated cytotoxic
immune cells have been genetically modified to be resistant to a
therapeutic agent; administering to a patient in need thereof, an
effective amount of the therapeutic agent; administering to the
patient a population of isolated genetically modified cytotoxic
immune cells, whereupon the cytotoxic immune cells are delivered to
the tumor; and administering to the patient an effective amount of
an immune checkpoint inhibitor, thereby reducing the cancer in the
patient.
[0102] Preferably, the cytotoxic immune cells are .gamma..delta.
T-cells. Preferably the cytotoxic immune cells are .gamma..delta.
T-cells and natural killer (NK) cells. Preferably the cytotoxic
immune cells are .gamma..delta. T-cells, NK cells and may further
include other immunocompetent cells including, but not limited to:
monocytes, macrophages and dendritic cells. Preferably, the
cytotoxic immune cells can be isolated from the patient having the
cancer. Preferably, the cytotoxic immune cells may be isolated from
a source other than the patient with cancer.
[0103] Preferably, the cytotoxic immune cells are .gamma..delta.
T-cells derived from human induced pluripotent stem cells (hiPSCs).
Preferably, the cytotoxic immune cells are .gamma..delta. T-cells
and NK cells derived from human induced pluripotent stem cells
(hiPSCs). Preferably the cytotoxic immune cells are derived from
human induced pluripotent stem cells (hiPSCs) and are
.gamma..delta. T-cells, NK cells and may further include other
immunocompetent cells including, but not limited to: monocytes,
macrophages and dendritic cells. Preferably, the pluripotent stem
cells can be isolated from the patient having the cancer.
Preferably, the pluripotent stem cells may be isolated from a
source other than the patient with cancer.
[0104] Preferably, the therapeutic agent is characterized by a cell
developing resistance to said therapeutic agent when the cell
receives a heterologous nucleic acid, and wherein the heterologous
nucleic acid is expressed in the cell. Preferably, the therapeutic
agent can be a cytotoxic chemotherapeutic agent selected from the
group consisting of: alkylating agents (e.g., cyclophosphamide,
ifosfamide); metabolic antagonists (e.g., methotrexate (MTX),
5-fluorouracil or derivatives thereof); DNA demethylating agents
(also known as antimetabolites; e.g., azacitidine); a substituted
nucleotide; a substituted nucleoside; antitumor antibiotics (e.g.,
mitomycin, adriamycin); plant-derived antitumor agents (e.g.,
vincristine, vindesine, TAXOL.RTM., paclitaxel, abraxane);
cisplatin; carboplatin; etoposide; and the like. Such agents may
further include, but are not limited to, the anti-cancer agents
trimethotrexate (TMTX); temozolomide; raltitrexed;
S-(4-Nitrobenzyl)-6-thioinosine (NBMPR); 6-benzyguanidine (6-BG);
nitrosoureas [e.g., bis-chloronitrosourea (BCNU; carmustine),
lomustine (CCNU) +/-Procarbazine and Vincristine (PCV regimen),
fotemustine]; cytarabine; camptothecin; and a therapeutic
derivative of any thereof.
[0105] Preferably, the step of obtaining a population of cytotoxic
immune cells genetically modified to be resistant to a therapeutic
agent comprises: obtaining from a subject such as a human subject
or animal subject a population of cytotoxic immune cells, for
example by obtaining a biological sample from the subject including
but not limited to a blood or tissue sample including a tumor
biopsy. The sample may optionally be enriched for cytotoxic immune
cells and other immunocompetent cells and/or the cells present in
the sample may optionally be expanded to increase the population of
the cells present in the sample. The cells are preferably stably
transfected or gene edited with a vector comprising a heterologous
nucleic acid sequence operably linked to a promoter, wherein the
heterologous nucleic acid sequence encodes a polypeptide conferring
to the cell resistance to one or more therapeutic agents such as a
chemotherapeutic agent. Preferably, the population of stably
transfected cytotoxic immune cells can be viably maintained.
[0106] Preferably, the therapeutic agent can be trimethotrexate or
methotrexate, and the heterologous nucleic acid sequence encodes
dihydrofolate reductase, or a derivative thereof. Preferably, the
therapeutic agent can be temozolomide, or a therapeutically active
derivative thereof, and the heterologous nucleic acid sequence may
encode O.sup.6 methylguanine DNA methyltransferase, or a derivative
thereof. Preferably, the chemotherapeutic agent can be cisplatin,
doxorubicin or paclitaxel, or a therapeutically active derivative
thereof, and the heterologous nucleic acid sequence may encode
multiple drug resistance-1 protein (MDR1), or a derivative
thereof.
[0107] Preferably, the isolated genetically modified cytotoxic
immune cells, the therapeutic agent, and the immune checkpoint
inhibitor can be co-administered to the patient. Preferably, the
genetically modified cytotoxic immune cells, the therapeutic agent,
and the immune checkpoint inhibitor can be sequentially
administered to the patient. Preferably, the genetically modified
cytotoxic immune cells, the therapeutic agent, and the immune
checkpoint inhibitor can be concomitantly administered to the
patient. Preferably, the genetically modified cytotoxic immune
cells are administered to the patient directly into the tumor or to
a blood vessel proximal and leading into the tumor. Preferably, the
tumor is a glioblastoma.
[0108] Preferably, the immune checkpoint inhibitor can be a
biologic therapeutic or a small molecule. Preferably, the
checkpoint inhibitor is a monoclonal antibody, a humanized
antibody, a fully human antibody, a fusion protein, an
antigen-binding fragment or a combination thereof. Preferably, the
checkpoint inhibitor inhibits a checkpoint protein which may be
CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9,
LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, OX40,
B-7 family ligands or a combination thereof. Preferably, the
checkpoint inhibitor interacts with a ligand of a checkpoint
protein which may be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA,
HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1,
CHK2, A2aR, OX40, B-7 family ligands or a combination thereof.
Illustrative immune checkpoint inhibitors include Tremelimumab
(CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal Antibody
(Anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker),
OPDIVO.RTM./Nivolumab (anti-PD1 antibody), CT-011 (anti-PD1
antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody),
BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody),
MSB0010718C (anti-PDL1 antibody) and YERVOY.RTM./ipilimumab
(anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands
include, but are not limited to PD-L1, PD-L2, B7-H3, B7-H4, CD28,
CD86 and TIM-3.
[0109] Preferably, the present invention provides the use of a
class of immune checkpoint inhibitor drugs that inhibit CTLA-4.
Suitable anti-CTLA4 antagonist agents for use in the methods of the
invention, include, without limitation, anti-CTLA4 antibodies,
human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian
anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, monoclonal
anti-CTLA4 antibodies, polyclonal anti-CTLA4 antibodies, chimeric
anti-CTLA4 antibodies, MDX-010 (ipilimumab), tremelimumab,
anti-CD28 antibodies, anti-CTLA4 adnectins, anti-CTLA4 domain
antibodies, single chain anti-CTLA4 fragments, heavy chain
anti-CTLA4 fragments, light chain anti-CTLA4 fragments, and
inhibitors of CTLA4 that agonize the co-stimulatory pathway.
Preferably, additional anti-CTLA4 antagonists include, but are not
limited to, the following: any inhibitor that is capable of
disrupting the ability of CD28 antigen to bind to its cognate
ligand, to inhibit the ability of CTLA4 to bind to its cognate
ligand, to augment T cell responses via the co-stimulatory pathway,
to disrupt the ability of B7 to bind to CD28 and/or CTLA4, to
disrupt the ability of B7 to activate the co-stimulatory pathway,
to disrupt the ability of CD80 to bind to CD28 and/or CTLA4, to
disrupt the ability of CD80 to activate the co-stimulatory pathway,
to disrupt the ability of CD86 to bind to CD28 and/or CTLA4, to
disrupt the ability of CD86 to activate the co-stimulatory pathway,
and to disrupt the co-stimulatory pathway, in general from being
activated. This necessarily includes small molecule inhibitors of
CD28, CD80, CD86, CTLA4, among other members of the co-stimulatory
pathway; antibodies directed to CD28, CD80, CD86, CTLA4, among
other members of the co-stimulatory pathway; antisense molecules
directed against CD28, CD80, CD86, CTLA4, among other members of
the co-stimulatory pathway; adnectins directed against CD28, CD80,
CD86, CTLA4, among other members of the co-stimulatory pathway,
RNAi inhibitors (both single and double stranded) of CD28, CD80,
CD86, CTLA4, among other members of the co-stimulatory pathway,
among other anti-CTLA4 antagonists.
[0110] Preferably, immunostimulatory agents, T cell growth factors
and interleukins may be used in combination with checkpoint
inhibitors and drug-resistant immunotherapy. Immunostimulatory
agents are substances (drugs and nutrients) that stimulate the
immune system by inducing activation or increasing activity of any
of its components. Immunostimulants include, but are not limited
to, bacterial vaccines, colony stimulating factors, interferons,
interleukins, other immunostimulants, therapeutic vaccines, vaccine
combinations and viral vaccines. T cell growth factors are proteins
that stimulate the proliferation of T cells. Examples of T cell
growth factors include Il-2, IL-7, IL-15, IL-17, IL21 and
IL-33.
[0111] Preferably the invention provides compositions comprising at
least one checkpoint inhibitor, and an isolated population of
cytotoxic immune cells comprising .gamma..delta. T-cells, NK cells,
or any combination thereof wherein greater than about 5% and
preferably greater than about 50% of the population of cytotoxic
immune cells expresses a polypeptide that confers resistance to a
chemotherapy agent. Preferably the composition comprises at least
one checkpoint inhibitor, and an isolated population of cytotoxic
immune cells comprising .gamma..delta. T-cells and NK cells wherein
about 50% to about 95% of the population of cytotoxic immune cells
are .gamma..delta. T-cells and wherein about 5% to about 25% of the
population of cytotoxic immune cells are NK cells.
[0112] Preferably, the therapeutic, immune checkpoint inhibitor,
biologic therapeutic or pharmaceutical composition as disclosed
herein can be administered to an individual by various routes
including, for example, orally or parenterally, such as
intravenously, intramuscularly, subcutaneously, intraorbitally,
intracapsularly, intraperitoneally, intrarectally,
intracisternally, intratumorally, intravasally, intradermally or by
passive or facilitated absorption through the skin using, for
example, a skin patch or transdermal iontophoresis, respectively.
The therapeutic, checkpoint inhibitor, biologic therapeutic or
pharmaceutical composition also can be administered to the site of
a pathologic condition, for example, intravenously or
intra-arterially into a blood vessel supplying a tumor.
[0113] Preferably, the immune checkpoint inhibitor is administered
in less than 0.0001 mg/kg, 0.0001-0.001 mg/kg, 0.001-0.01 mg/kg,
0.01-0.05 mg/kg, 0.05-0.1 mg/kg, 0.1-0.2 mg/kg, 0.2-0.3 mg/kg,
0.3-0.5 mg/kg, 0.5-0.7 mg/kg, 0.7-1 mg/kg, 1-2 mg/kg, 2-3 mg/kg,
3-4 mg/kg, 4-5 mg/kg, 5-6 mg/kg, 6-7 mg/kg, 7-8 mg/kg, 8-9 mg/kg,
9-10 mg/kg, at least 10 mg/kg, or any combination thereof doses.
Preferably, the checkpoint inhibitor is administered at least once
a week, at least twice a week, at least three times a week, at
least once every two weeks, or at least once every month or
multiple months. Preferably, the checkpoint inhibitor is
administered as a single dose, in two doses, in three doses, in
four doses, in five doses, or in 6 or more doses.
[0114] Preferably, the invention provides systems for treating a
cancer in a patient comprising a cytotoxic therapeutic agent having
the characteristics of inhibiting the survival of a cancer cell, an
isolated population of cytotoxic immune cells, where the cytotoxic
immune cells genetically modified to be resistant to the
therapeutic agent, and an immune checkpoint inhibitor. Preferably,
the population of cytotoxic immune cells comprises .gamma..delta.
T-cells, NK cells or any combination thereof. Preferably the
population of cytotoxic immune cells comprises .gamma..delta.
T-cells and natural killer cells and optionally comprises other
immunocompetent cells. Preferably, the population of cytotoxic
immune cells may comprise a heterologous nucleic acid sequence
operably linked to a promoter, where the heterologous nucleic acid
sequence encodes a polypeptide that when expressed in a cell
confers resistance to the therapeutic agent to the cell.
Preferably, the therapeutic agent is a cytotoxic chemotherapeutic
agent selected from the group consisting of: an alkylating agent
(e.g., cyclophosphamide, ifosfamide); a metabolic antagonist (e.g.,
methotrexate (MTX), 5-fluorouracil or derivatives thereof); a DNA
demethylating agent (also known as antimetabolites; e.g.,
azacitidine); a substituted nucleotide; a substituted nucleoside;
an antitumor antibiotic (e.g., mitomycin, adriamycin); a
plant-derived antitumor agent (e.g., vincristine, vindesine,
TAXOL.RTM., paclitaxel, abraxane); cisplatin; carboplatin;
etoposide; and the like. Such agents may further include, but are
not limited to, the anti-cancer agents trimethotrexate (TMTX);
temozolomide; raltitrexed; S-(4-Nitrobenzyl)-6-thioinosine (NBMPR);
6-benzyguanidine (6-BG); nitrosoureas [e.g., bis-chloronitrosourea
(BCNU; carmustine), lomustine (CCNU) +/-Procarbazine and
Vincristine (PCV regimen), fotemustine]; cytarabine; camptothecin;
and a therapeutic derivative of any thereof. Preferably, the
therapeutic agent is trimethotrexate or methotrexate, and the
heterologous nucleic acid sequence encodes dihydrofolate reductase,
or a derivative thereof. Preferably, the therapeutic agent is
temozolomide, or a therapeutically agent derivative thereof, and
the heterologous nucleic acid sequence encodes O.sup.6
methylguanine DNA methyltransferase (MGMT), or a derivative
thereof. Preferably, the therapeutic agent is a nitrosourea, or a
therapeutically agent derivative thereof, and the heterologous
nucleic acid sequence encodes O.sup.6 methylguanine DNA
methyltransferase (MGMT), or a derivative thereof.
[0115] Preferably, the immune checkpoint inhibitor is a biologic
therapeutic or a small molecule. Preferably, the checkpoint
inhibitor is a monoclonal antibody, a humanized antibody, a fully
human antibody, a fusion protein, an antigen-binding fragment or a
combination thereof. Preferably, the checkpoint inhibitor inhibits
a checkpoint protein which may be CTLA-4, PDL1, PDL2, PD1, B7-H3,
B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160,
CGEN-15049, CHK 1, CHK2, A2aR, OX40, B-7 family ligands or a
combination thereof. Preferably, the checkpoint inhibitor interacts
with a ligand of a checkpoint protein which may be CTLA-4, PDL1,
PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR,
2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, OX40, B-7 family ligands
or a combination thereof. Illustrative immune checkpoint inhibitors
include Tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1
monoclonal Antibody (Anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker),
OPDIVO.RTM./Nivolumab (anti-PD1 antibody), CT-011 (anti-PD1
antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody),
BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody),
MSB0010718C (anti-PDL1 antibody) and YERVOY.RTM./ipilimumab
(anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands
include, but are not limited to PD-L1, PD-L2, B7-H3, B7-H4, CD28,
CD86 and TIM-3.
[0116] Preferably, the present invention covers the use of a class
of checkpoint inhibitor drugs that inhibit CTLA-4. Suitable
anti-CTLA4 antagonist agents for use in the methods of the
invention, include, without limitation, anti-CTLA4 antibodies,
human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian
anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, monoclonal
anti-CTLA4 antibodies, polyclonal anti-CTLA4 antibodies, chimeric
anti-CTLA4 antibodies, MDX-010 (ipilimumab), tremelimumab,
anti-CD28 antibodies, anti-CTLA4 adnectins, anti-CTLA4 domain
antibodies, single chain anti-CTLA4 fragments, heavy chain
anti-CTLA4 fragments, light chain anti-CTLA4 fragments, and
inhibitors of CTLA4 that agonize the co-stimulatory pathway.
[0117] Preferably, additional anti-CTLA4 antagonists include, but
are not limited to, the following: any inhibitor that is capable of
disrupting the ability of CD28 antigen to bind to its cognate
ligand, to inhibit the ability of CTLA4 to bind to its cognate
ligand, to augment T cell responses via the co-stimulatory pathway,
to disrupt the ability of B7 to bind to CD28 and/or CTLA4, to
disrupt the ability of B7 to activate the co-stimulatory pathway,
to disrupt the ability of CD80 to bind to CD28 and/or CTLA4, to
disrupt the ability of CD80 to activate the co-stimulatory pathway,
to disrupt the ability of CD86 to bind to CD28 and/or CTLA4, to
disrupt the ability of CD86 to activate the co-stimulatory pathway,
and to disrupt the co-stimulatory pathway, in general from being
activated. This necessarily includes small molecule inhibitors of
CD28, CD80, CD86, CTLA4, among other members of the co-stimulatory
pathway; antibodies directed to CD28, CD80, CD86, CTLA4, among
other members of the co-stimulatory pathway; antisense molecules
directed against CD28, CD80, CD86, CTLA4, among other members of
the co-stimulatory pathway; adnectins directed against CD28, CD80,
CD86, CTLA4, among other members of the co-stimulatory pathway,
RNAi inhibitors (both single and double stranded) of CD28, CD80,
CD86, CTLA4, among other members of the co-stimulatory pathway,
among other anti-CTLA4 antagonists.
[0118] Preferably, immunostimulatory agents, T cell growth factors
and interleukins may be used. Immunostimulatory agents are
substances (drugs and nutrients) that stimulate the immune system
by inducing activation or increasing activity of any of its
components. Immunostimulants include, but are not limited to,
bacterial vaccines, colony stimulating factors, interferons,
interleukins, other immunostimulants, therapeutic vaccines, vaccine
combinations and viral vaccines. T cell growth factors are proteins
that stimulate the proliferation of T cells. Examples of T cell
growth factors include Il-2, IL-7, IL-15, IL-17, IL21 and
IL-33.
[0119] Preferably, the therapeutic, checkpoint inhibitor, biologic
therapeutic or pharmaceutical composition as disclosed herein can
be administered to an individual by various routes including, for
example, orally or parenterally, such as intravenously,
intramuscularly, subcutaneously, intraorbitally, intracapsularly,
intraperitoneally, intrarectally, intracisternally, intratumorally,
intravasally, intradermally or by passive or facilitated absorption
through the skin using, for example, a skin patch or transdermal
iontophoresis, respectively. The therapeutic, checkpoint inhibitor,
biologic therapeutic or pharmaceutical composition also can be
administered to the site of a pathologic condition, for example,
intravenously or intra-arterially into a blood vessel supplying a
tumor.
[0120] Preferably, the total amount of an agent to be administered
in practicing a method of the invention can be administered to a
subject as a single dose, either as a bolus or by infusion over a
relatively short period of time, or can be administered using a
fractionated treatment protocol, in which multiple doses are
administered over a prolonged period of time. One skilled in the
art would know that the amount of the composition to treat a
pathologic condition in a subject depends on many factors including
the age and general health of the subject as well as the route of
administration and the number of treatments to be administered. In
view of these factors, the skilled artisan would adjust the
particular dose as necessary.
[0121] Preferably, the checkpoint inhibitor is administered in less
than 0.0001 mg/kg, 0.0001-0.001 mg/kg, 0.001-0.01 mg/kg, 0.01-0.05
mg/kg, 0.05-0.1 mg/kg, 0.1-0.2 mg/kg, 0.2-0.3 mg/kg, 0.3-0.5 mg/kg,
0.5-0.7 mg/kg, 0.7-1 mg/kg, 1-2 mg/kg, 2-3 mg/kg, 3-4 mg/kg, 4-5
mg/kg, 5-6 mg/kg, 6-7 mg/kg, 7-8 mg/kg, 8-9 mg/kg, 9-10 mg/kg, at
least 10 mg/kg, or any combination thereof doses. Preferably, the
checkpoint inhibitor is administered at least once a week, at least
twice a week, at least three times a week, at least once every two
weeks, or at least once every month or multiple months. Preferably,
the checkpoint inhibitor is administered as a single dose, in two
doses, in three doses, in four doses, in five doses, or in 6 or
more doses.
[0122] Preferably, the invention provides systems for treating a
glioblastoma in a patient comprising a therapeutic agent having the
characteristics of inhibiting the survival of a cancer cell and
inducing a stress protein in the cancer cell, an isolated
population of cytotoxic immune cells, wherein said cytotoxic immune
cells comprise .gamma..delta. T-cells, NK cells, or any combination
thereof and further optionally comprise other immunocompetent cells
wherein the population of cytotoxic immune cells have been
genetically modified to be resistant to the therapeutic agent, and
a checkpoint inhibitor. Preferably, the population cytotoxic immune
cells comprise a heterologous nucleic acid sequence operably linked
to a promoter, wherein the heterologous nucleic acid sequence
encodes a polypeptide that when expressed in a cell confers
resistance to the therapeutic agent to the cell.
[0123] Preferably, the therapeutic agent is a cytotoxic
chemotherapeutic agent selected from the group consisting of: an
alkylating agent, a metabolic antagonist, an antitumor antibiotic,
and a plant-derived antitumor agent.
[0124] Preferably, the therapeutic agent is selected from the group
consisting of: trimethotrexate (TMTX), methotrexate (MTX),
temozolomide, reltritrexed, S-(4-Nitrobenzyl)-6-thioinosine
(NBMPR), camptothecin, 6-benzylguanidine, cytarabine, and a
therapeutic derivative of any thereof.
[0125] Preferably, the therapeutic agent is trimethotrexate or
methotrexate, and the heterologous nucleic acid sequence encodes
dihydrofolate reductase, or a derivative thereof.
[0126] Preferably, the immune checkpoint inhibitor can be a
biologic therapeutic or a small molecule. Preferably, the
checkpoint inhibitor is a monoclonal antibody, a humanized
antibody, a fully human antibody, a fusion protein, an
antigen-binding fragment or a combination thereof. Preferably, the
checkpoint inhibitor inhibits a checkpoint protein which may be
CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9,
LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, OX40,
B-7 family ligands or a combination thereof.
[0127] Preferably, the checkpoint inhibitor interacts with a ligand
of a checkpoint protein which may be CTLA-4, PDL1, PDL2, PD1,
B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160,
CGEN-15049, CHK 1, CHK2, A2aR, OX40, B-7 family ligands or a
combination thereof. Illustrative immune checkpoint inhibitors
include Tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1
monoclonal Antibody (Anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker),
OPDIVO.RTM./Nivolumab (anti-PD1 antibody), CT-011 (anti-PD1
antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody),
BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody),
MSB0010718C (anti-PDL1 antibody) and YERVOY.RTM./ipilimumab
(anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands
include, but are not limited to PD-L1, PD-L2, B7-H3, B7-H4, CD28,
CD86 and TIM-3.
[0128] Preferably, the present invention covers the use of a
specific class of immune checkpoint inhibitor drugs that inhibit
CTLA-4. Suitable anti-CTLA4 antagonist agents for use in the
methods of the invention, include, without limitation, anti-CTLA4
antibodies, human anti-CTLA4 antibodies, mouse anti-CTLA4
antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4
antibodies, monoclonal anti-CTLA4 antibodies, polyclonal anti-CTLA4
antibodies, chimeric anti-CTLA4 antibodies, MDX-010 (ipilimumab),
tremelimumab, anti-CD28 antibodies, anti-CTLA4 adnectins,
anti-CTLA4 domain antibodies, single chain anti-CTLA4 fragments,
heavy chain anti-CTLA4 fragments, light chain anti-CTLA4 fragments,
and inhibitors of CTLA4 that agonize the co-stimulatory
pathway.
[0129] Preferably, additional anti-CTLA4 antagonists include, but
are not limited to, the following: any inhibitor that is capable of
disrupting the ability of CD28 antigen to bind to its cognate
ligand, to inhibit the ability of CTLA4 to bind to its cognate
ligand, to augment T cell responses via the co-stimulatory pathway,
to disrupt the ability of B7 to bind to CD28 and/or CTLA4, to
disrupt the ability of B7 to activate the co-stimulatory pathway,
to disrupt the ability of CD80 to bind to CD28 and/or CTLA4, to
disrupt the ability of CD80 to activate the co-stimulatory pathway,
to disrupt the ability of CD86 to bind to CD28 and/or CTLA4, to
disrupt the ability of CD86 to activate the co-stimulatory pathway,
and to disrupt the co-stimulatory pathway, in general from being
activated. This necessarily includes small molecule inhibitors of
CD28, CD80, CD86, CTLA4, among other members of the co-stimulatory
pathway; antibodies directed to CD28, CD80, CD86, CTLA4, among
other members of the co-stimulatory pathway; antisense molecules
directed against CD28, CD80, CD86, CTLA4, among other members of
the co-stimulatory pathway; adnectins directed against CD28, CD80,
CD86, CTLA4, among other members of the co-stimulatory pathway,
RNAi inhibitors (both single and double stranded) of CD28, CD80,
CD86, CTLA4, among other members of the co-stimulatory pathway,
among other anti-CTLA4 antagonists.
[0130] Preferably, immunostimulatory agents, T cell growth factors
and interleukins may be used. Immunostimulatory agents are
substances (drugs and nutrients) that stimulate the immune system
by inducing activation or increasing activity of any of its
components. Immunostimulants include, but are not limited to,
bacterial vaccines, colony stimulating factors, interferons,
interleukins, other immunostimulants, therapeutic vaccines, vaccine
combinations and viral vaccines. T cell growth factors are proteins
that stimulate the proliferation of T cells. Examples of T cell
growth factors include Il-2, IL-7, IL-15, IL-17, IL21 and
IL-33.
[0131] Preferably, the therapeutic, immune checkpoint inhibitor,
biologic therapeutic or pharmaceutical composition as disclosed
herein can be administered to an individual by various routes
including, for example, orally or parenterally, such as
intravenously, intramuscularly, subcutaneously, intraorbitally,
intracapsularly, intraperitoneally, intrarectally,
intracisternally, intratumorally, intravasally, intradermally or by
passive or facilitated absorption through the skin using, for
example, a skin patch or transdermal iontophoresis, respectively.
The therapeutic, checkpoint inhibitor, biologic therapeutic or
pharmaceutical composition also can be administered to the site of
a pathologic condition, for example, intravenously or
intra-arterially into a blood vessel supplying a tumor.
[0132] Preferably, the total amount of an agent to be administered
in practicing a method of the invention can be administered to a
subject as a single dose, either as a bolus or by infusion over a
relatively short period of time, or can be administered using a
fractionated treatment protocol, in which multiple doses are
administered over a prolonged period of time. One skilled in the
art would know that the amount of the composition to treat a
pathologic condition in a subject depends on many factors including
the age and general health of the subject as well as the route of
administration and the number of treatments to be administered. In
view of these factors, the skilled artisan would adjust the
particular dose as necessary.
[0133] Preferably, the checkpoint inhibitor is administered in less
than 0.0001 mg/kg, 0.0001-0.001 mg/kg, 0.001-0.01 mg/kg, 0.01-0.05
mg/kg, 0.05-0.1 mg/kg, 0.1-0.2 mg/kg, 0.2-0.3 mg/kg, 0.3-0.5 mg/kg,
0.5-0.7 mg/kg, 0.7-1 mg/kg, 1-2 mg/kg, 2-3 mg/kg, 3-4 mg/kg, 4-5
mg/kg, 5-6 mg/kg, 6-7 mg/kg, 7-8 mg/kg, 8-9 mg/kg, 9-10 mg/kg, at
least 10 mg/kg, or any combination thereof doses. Preferably, the
checkpoint inhibitor is administered at least once a week, at least
twice a week, at least three times a week, at least once every two
weeks, or at least once every month or multiple months. Preferably,
the checkpoint inhibitor is administered as a single dose, in two
doses, in three doses, in four doses, in five doses, or in 6 or
more doses.
[0134] It should be noted that ratios, concentrations, amounts, and
other numerical data may be expressed herein in a range format. It
is to be understood that such a range format is used for
convenience and brevity, and thus, should be interpreted in a
flexible manner to include not only the numerical values explicitly
recited as the limits of the range, but also to include all the
individual numerical values or sub-ranges encompassed within that
range as if each numerical value and sub-range is explicitly
recited. To illustrate, a concentration range of "about 0.1% to
about 5%" should be interpreted to include not only the explicitly
recited concentration of about 0.1 wt. % to about 5 wt. %, but also
include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and
the sub-ranges (e.g., 0.5% , 1.1%, 2.2%, 3.3%, and 4.4%) within the
indicated range. The term "about" can include .+-.1%, .+-.2% ,
.+-.3%, .+-.4%, .+-.5%, .+-.6%, .+-.7%, .+-.8%, .+-.9%, or .+-.10%,
or more of the numerical value(s) being modified. In addition, the
phrase "about `x` to `y`" includes "about `x` to about `y`".
[0135] Many variations and modifications may be made to the
above-described invention without departing substantially from the
spirit and principles of the invention. All such modifications and
variations are intended to be included herein within the scope of
this invention and protected by the following claims.
EXAMPLES
[0136] The following examples are offered by way of illustration
and are not to be construed as limiting the invention as claimed in
any way.
Example 1
[0137] Cancer Treatment Paradigms
[0138] A combination therapy comprising a chemotherapeutic agent,
drug-resistant .gamma..delta.--T cells and/or drug-resistant
natural killer cells, and an immune checkpoint inhibitor(s) is used
to treat the cancer patient. The chemotherapeutic agent is selected
from alkylating agents (e.g., cyclophosphamide, ifosfamide),
metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil or
derivatives thereof), antitumor antibiotics (e.g., mitomycin,
adriamycin), plant-derived antitumor agents (e.g., vincristine,
vindesine, TAXOL.RTM., paclitaxel, abraxane), cisplatin,
carboplatin, etoposide, and the like. Such agents may further
include, but are not limited to, the anti-cancer agents
trimethotrexate (TMTX), temozolomide, raltitrexed,
S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), 6-benzyguanidine (6-BG),
bis-chloronitrosourea (BCNU), cytarabine, and camptothecin, or a
therapeutic derivative of any thereof. The drug-resistant
.gamma..delta.--T cells and natural killer cells are resistant to
the chemotherapeutic agent. The immune checkpoint inhibitor(s) is
used alone or in combination with other immune checkpoint
inhibitors, and is selected from inhibitors of the immune
checkpoint proteins CTLA-4, PDL1 (B7-H1, CD274), PDL2 (B7-DC,
CD273), PD1, B7-H3 (CD276), B7-H4 (B7-S1, B7x, VCTN1), BTLA
(CD272), HVEM, TIM3 (HAVcr2), GAL9, LAG3 (CD223), VISTA, KIR, 2B4
(CD244; belongs to the CD2 family of molecules and is expressed on
all NK, .gamma..delta., and memory CD8.sup.+ (.alpha..beta.) T
cells), CD160 (also referred to as BY55), CGEN-15049, CHK 1 and
CHK2 kinases, OX40, A2aR and various B-7 family ligands.
Evaluation of Immune Therapy Activity in Combination with
Chemotherapy
[0139] Response assessment criteria include evaluation based upon
Response Evaluation Criteria in Solid Tumors (WHO; World Health
Organization Offset Publication No. 48, 1979); RECIST (Response
Evaluation Criteria in Solid Tumors; J Natl Cancer Inst 2000;
92:205-16; Eisenhauer EA et al., Eur J Cancer 2009;45:228-47);
Immune-Related Response Criteria (Jedd W E et al., Clin Cancer Res
2009;15(23),7412-7420); and iRANO (Immunotherapy Response
Assessment in Neuro-Oncology; Okada H et al., Lancet Oncol 2015
November; 16(15):e534-42).
Results
[0140] Chemotherapy as a combination therapy additionally
comprising drug-resistant .gamma..delta.--T cells (and/or
drug-resistant natural killer cells) and an inhibitor(s) of immune
checkpoint proteins results in an enhanced or prolonged anti-tumor
response; a medically beneficial response; additive or synergistic
antitumor activity, when compared with chemotherapy alone, or
chemotherapy combined with drug-resistant .gamma..delta.--T cells
(and/or drug-resistant natural killer cells); shrinkage in baseline
lesions, without new lesions; tumor regression; durable stable
disease with possible steady decline in total tumor burden;
response after an increase in total tumor burden; and response in
the presence of new lesions. Other clinical findings may include an
increase in tumor infiltrating lymphocytes as measured by an
increase in tumor size; and long-term survival promotion.
[0141] For example, temozolomide (TMZ)-treated tumors express
increased numbers of stress ligands on the surface of the tumor
cell, such as MICA, MICB, and ULBP1-6. The killing of tumor cells
is facilitated by the drug-resistant .gamma..delta.--T cells and/or
drug-resistant natural killer cells whose receptor(s), such as
NKG2D, recognizes the tumor-specific stress ligands. However, tumor
cells can enzymatically cleave MICA and MICB, releasing soluble
MICA and MICB. When bound by effector cell receptors in the plasma,
this can lead to down regulation of the receptor expression
resulting in immune escape. Chemotherapies such as TMZ have been
found to increase cell surface expression of stress ligands on the
tumor tissue without significantly increasing the concentration of
the soluble ligands in the plasma. Responders to antibody
inhibitors of CTLA-4 have demonstrated the ability to produce a
humoral response against NKG2D ligands such as MICA. These
responses result in a reduction in soluble MICA, which may allow
the amplified stress ligand expression on the tumor surface to
enhance tumor cell killing by the drug-resistant .gamma..delta.--T
cells and/or drug-resistant natural killer cells.
Example 2
[0142] Analysis of Checkpoint Molecule Expression and Function in
Glioma and .gamma..delta. T Cells
[0143] Checkpoint molecule expression on GMP manufactured
.gamma..delta. T cells and glioblastoma tumor cells was evaluated
and the function of enriched .gamma..delta. T cells in-vitro was
assessed.
Methods
[0144] Ex Vivo Expansion of .gamma..delta. T Cells
[0145] Human apheresis products collection from healthy donors were
purchased from Hemacare (Van Nuys Calif.). The products were
transferred to the UAB Cell Therapy Laboratory GMP facility. For
static culture: All manipulations were performed in a Class 100
laminar flow hood surrounded by a Class 10K/ISO 7 classified air
flow. The product was diluted v/v with HBSS (Hank's Balanced Salt
Solution) and mononuclear cells isolated by density gradient
separation on Ficoll (Sigma-Aldrich; St. Louis, Mo.). Interphase
was harvested and washed X2 in HBSS. The cell pellet was
resuspended in CTS.TM. OpTmizer.TM. T Cell Expansion serum free
media (ThermoFisher Scientific; Waltham, Mass.) supplemented with 2
mM Zoledronic Acid (Novartis; Basel HV) and 100u/mL IL2 (Miltenyi
Biotec Ltd; Bergisch Gladbach, Germany). Cells were cultured in
standard T150 or T75 flasks. For closed system culture: All
operation including preparation of media, stocks and buffers were
performed in ISO 7 clean rooms under biosafety cabinets using GMP
grade reagents. Automated Ficoll separation of the product and
cultivation were carried out in CLINIMACS PRODIGY.RTM. (Miltenyi
Biotec Ltd; Bergisch Gladbach, Germany) bioreactor using customized
cell processing program and TS520 tubing set. Cells were then
expanded in the CentriCult-Unit of the CLINIMACS PRODIGY.RTM. using
a combination of Zoledronate (Zometa; Novartis, Basel) and
Interleukin-2 (Miltenyi Biotec Ltd; Bergisch Gladbach, Germany)
(ZOL/IL-2), in OpTmizer serum-free culture (Thermo Fisher
Scientific; Waltham, Mass.). The culture was kept at a density of
1-2.times.106 cells/ml and supplemented with IL-2 100u/mL every
other day until cells harvest at day 13. The process was
continuously monitored to determine kinetics of expansion and
phenotype in comparison with small-scale culture in flasks at
regular intervals. Following 13-day culture, expanded
.gamma..delta. T cells were phenotyped and cytotoxicity determined
against standard leukemia cell lines and glioma tumor cells.
Flow Cytometry Analysis
[0146] Multiparametric flow cytometry analysis was used to
determine phenotype & functional profile in donor PBMC and
cultured products. DuraClone T cell subset (Beckman Coulter) plus
anti-PD-1, anti-CTLA-4 and anti-PD-L1 panel were used to monitor
expression of checkpoint molecule and .gamma..delta. T cell
enrichment using Flow cytometry. Samples of product were analyzed
on culture day 1 and 13. Freshly dissociated PDX derived
glioblastoma tumor cells were analyzed for PD-L1 expression using
flow cytometry. Single cell suspension of PDX derived glioblastoma
cells were blocked with FcR Block (Biolegend) for 15 min, before
staining with PD-L1 antibody and respective Isotype (IgG1). The
stained cells were acquired on BD fortessa X-20 flow cytometer.
Flow Cytometry Based Cytotoxicity Evaluation
[0147] Potency of the cell product was determined using in-vitro
cytotoxicity assays against K562 cells. Expanded .gamma..delta. T
cells was assessed at day 13 for cytotoxicity on human leukemic
cell lines (K562), and tumor cells (JX22T, JX12T AND JX59T) using
flow cytometry based cytotoxicity assay. The Basic Cytotoxicity
Assay Kit (https://immunochemistry.com) includes two fluorescent
reagents: CFSE and 7-AAD. CFSE, a green fluorescing membrane stain
was used to label the target cells (K562). The unstained effector
cells are added at increasing effector to target ratio and
incubated with the target cells for four hours before adding 7-AAD,
a red fluorescing live/dead stain followed by acquisition and
analyzes using flow cytometer. The Cytotoxicity is calculated as
the percentage of the green target cells which are detected red
channel. Formula for % Cytotoxicity=# Dead cells/# Live cells +#
Dead cells* 100.
Results & Discussion
[0148] Freshly isolated PBMCs from two healthy donors (donor 1:
043692; donor 2: 043988) were used for expansion of .gamma..delta.
T cells in vitro. Upon examining checkpoint expression by flow
cytometry, we noted upregulation of PD-1 and CTLA-4 and PD-L1 in
expanded .gamma..delta. T cells. On day 13, PD-1 increase more than
20%, CTLA-4 expression ranged from 3 to 6%. Interestingly, PD-L1
was also upregulated (>9%) in both donors, [FIGS. 1-4]. No
significant change in levels of expression of checkpoint molecules
in MGMT transduced .gamma..delta. T cells as compared to
untransduced .gamma..delta. T cells was seen. Moderate
downregulation of PD-1 and CTLA-4 on resting (no IL2 and ZOL)
.gamma..delta. T cells when compared to stimulated cultures was
also observed [FIG. 5].
[0149] PD-L1 expression on PDX derived Glioma cells was also
analyzed by flow cytometry. As compared to isotype controls, PD-L1
receptor expression was low or undetectable. The percentage of
PD-L1 expression varied from JX12T (0%), JX22T (1.8%) and JX59T
(10.4%) [FIG. 6].
[0150] Next, we examined whether the blockade of immune inhibitory
receptors had any effect on .gamma..delta. T cells ability to
increase cytotoxicity to cancer cells [FIG. 7]. Glioma tumor cells
(JX22T, JX12T, JX59T) and K562 (a cell line established from
chronic myelogenous leukemia) were challenged with manufactured
.gamma..delta. T cells at 20:1 ratio, in the assay mixture that
contained one or more checkpoint antibodies at a concentration of
20 .mu.g/ml. High level of cytolytic activity was observed for K562
[PD-L1 (52%), CTLA-4 (5%), CTLA-4+PD-1 (7%), PD-1 (7). Modest
increase in tumor cell lysis was observed for JX12T tumor cells for
PD-1 (3%), CTLA-4 (2) PD-1+ CTLA-4 (5) combo and PD-L1 (8)
blockade. However, checkpoint blockade did not increase the
cytotoxicity of MGMT modified .gamma..delta. T cells for JX22T AND
JX59T.
[0151] Our observation that glioblastoma tumor cells does not
express significant levels of PD-L1 is in concordance with already
published reports, by Garg et al. [Preclinical efficacy of
immune-checkpoint monotherapy does not recapitulate corresponding
biomarkers-based clinical predictions in glioblastoma.
OncoImmunology, 2016]. A study lead by Joseph et al. found high
level of PD-L1 expression in tumor infiltrating myeloid cells in
the glioblastoma tumors [Immunosuppressive tumor-infiltrating
myeloid cells mediate adaptive immune resistance via a PD-1/PD-L1
mechanism in glioblastoma, Neuro-Oncology 9(6), 796-806, 2017].
Since, myeloid/stromal compartment in the glioma tumor
microenvironment express high levels of PD-L1 and our observation
that GMP manufactured T cells/.gamma..delta. T cells upregulates
PD-1, we propose a comprehensive treatment plan to include systemic
treatment using PD-1, PD-L1 blocking antibodies and Temozolomide
and localized infusion MGMT expressing .gamma..delta. T cells to
effectively treat glioblastoma and achieve high objective response
rate.
[0152] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
[0153] United States patent applications cited herein are
incorporated by reference. All published foreign patents and patent
applications cited herein are hereby incorporated by reference. All
other published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
[0154] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims. It will
also be understood that none of the embodiments described herein
are mutually exclusive and may be combined in various ways without
departing from the scope of the invention encompassed by the
appended claims.
* * * * *
References